CN104744591B

CN104744591B - Amyloid beta binding proteins

Info

Publication number: CN104744591B
Application number: CN201510136184.9A
Authority: CN
Inventors: S.巴格霍恩; H.希伦; A.施特里宾格; S.吉艾西; U.埃伯特; L.贝纳特伊尔
Original assignee: Abbott GmbH and Co KG; AbbVie Inc
Current assignee: Abbott GmbH and Co KG; AbbVie Inc
Priority date: 2010-04-15
Filing date: 2011-04-13
Publication date: 2022-09-27
Anticipated expiration: 2031-04-13
Also published as: WO2011130377A3; MX360403B; JP2016028030A; CN102933601B; MX336196B; US20180094049A1; JP2020103288A; US20150218261A1; US8987419B2; JP2018058837A; US20110256138A1; EP2558494B1; ES2684475T3; EP2558494A2; JP2013523182A; MX2012011934A; JP6691359B2; AR080914A1; US9822171B2; CA2796339C

Abstract

The present invention relates to amyloid-beta (a β) binding proteins. The antibodies of the invention have high affinity for the A β (20-42) globulomer or any A β form comprising globulomer epitopes. Methods of making and methods of using the antibodies of the invention are also provided.

Description

Amyloid beta binding proteins

The application is a divisional application of PCT application with the international application date of 2011, 4, 13, the international application number of PCT/US2011/032269, the application number of 201180029833.5 entering the national stage and the invention name of 'beta amyloid binding protein'.

Technical Field

The present invention relates to amyloid-beta (a β) binding proteins, nucleic acids encoding the proteins, methods of producing the proteins, compositions comprising the proteins, and uses of the proteins in the diagnosis, treatment, and prevention of conditions such as amyloidosis, e.g., alzheimer's disease.

Background

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive ability and characteristic neuropathological features including deposits of amyloid-beta (Abeta) peptide, neurofibrillary tangles and neuronal loss in several regions of the brain (Hardy and Selkoe, Science 297: 353, 2002; Mattson, Nature 431: 7004, 2004. cerebral amyloid deposits and cognitive impairment very similar to those observed in Alzheimer's disease are also hallmarks of Down syndrome (trisomy 21) which occurs at a frequency of about 1 birth in 800.

The a β peptide arises from the Amyloid Precursor Protein (APP) which is processed by proteolysis. This processing is achieved by the cooperative activity of several proteases, designated α -, β -and γ -secretases, and results in many specific fragments of varying length. Amyloid deposits are composed primarily of

peptides

40 or 42 amino acids in length (a β 40, a β 42). In addition to human variants, this also includes isoforms of amyloid beta (1-42) protein that are present in organisms other than humans, particularly in other mammals, especially rats. Such proteins that tend to polymerize in aqueous environments can exist in very different molecular forms. The simple association of the deposition of insoluble proteins with the appearance or progression of dementing disorders such as alzheimer's disease has proven to be unreliable. (Terry et al, Ann. neuron. 30: 572-19, 580, 1991; Dickson et al, neuron. Aging 16: 285-298, 1995). In contrast, loss of synaptic and cognitive perception appears to be better associated with the soluble form of A β (1-42) (Lue et al, Am. J. Pathol. 155: 853-862, 1999; McLean et al, Ann. neuron. 46: 860-866, 1999).

None of the polyclonal and monoclonal antibodies that have been raised in the past against monomeric a β (1-42) have proven to produce the desired therapeutic effect, nor have caused serious side effects in animals and/or humans. For example, passive immunization results from preclinical studies in the very old APP23 mouse, which received anti-a β (1-42) antibodies against the N-terminus once a week for 5 months, indicated therapeutically relevant side effects. In particular, these mice showed an increase in the number and severity of microhemorrhages compared to saline-treated mice (Pfeifer et al, Science 298: 1379, 2002). Similar increases in bleeding were also described for the very old (> 24 months) Tg2576 and PDAPP mice (Wilcock et al, J Neuroscience 23: 3745-51, 2003; Racke et al, J Neuroscience 25: 629-. In both lines, injection of anti-a β (1-42) resulted in a significant increase in microhemorrhages.

WO 2004/067561 relates to spherical oligomers ("globulomers") of a β (1-42) peptides and processes for preparing the same. WO 2006/094724 relates to non-diffusible spherical Α β (X-38.. 43) oligomers, wherein X is selected from the number 1.. 24. WO 2004/067561 and WO 2006/094724 further describe the restricted proteolysis of globulomers to obtain truncated forms of the globulomers, such as A β (20-42) or A β (12-42) globulomers. WO 2007/064917 describes the cloning, expression and isolation of recombinant forms of amyloid beta peptide (hereinafter referred to as N-Met A β (1-42)) and globulomer forms thereof. The data suggest the existence of an amyloid fibril independent pathway for a β folding and assembly into a β globulomers that display one or more unique epitopes (hereinafter referred to as globulomer epitopes). Since globulomer epitopes were detected in the brains of AD patients and APP transgenic mice, and globulomers specifically bind neurons and block LTP, globulomers represent pathologically relevant a β conformers. Soluble a β globulomers have been found to exert their deleterious effects essentially through interaction with P/Q type presynaptic calcium channels, and inhibitors of this interaction are therefore useful for the treatment of amyloidosis, such as alzheimer's disease (WO 2008/104385).

Antibodies that selectively bind to such globulomeric forms of a β have been described in WO 2007/064972, WO 2007/062852, WO 2008067464, WO 2008/150946 and WO 2008/150949. For example, several monoclonal antibodies known from WO 2007/062852 and WO 2008/150949 specifically recognize the A β (20-42) globulomer.

There is a great, unmet therapeutic need for the development of biologics such as a β binding proteins that prevent or slow the progression of disease without inducing negative and potentially lethal effects on the human body. Such a need is particularly apparent in view of the increasing lifespan of the general population, and the concomitant increase in the number of patients diagnosed with alzheimer's disease or related disorders annually with such an increase. Further, such a β binding proteins would allow for a correct diagnosis of alzheimer's disease in patients experiencing its symptoms, a diagnosis that can only be confirmed after necropsy at present. In addition, a β binding proteins allow the elucidation of the biological properties of proteins and other biological factors responsible for this debilitating disease.

Disclosure of Invention

The present invention provides a novel family of a β binding proteins (or simply "binding proteins") capable of binding to soluble a β globulomers, such as a β (20-42) globulomers as described herein, CDR grafted antibodies, humanized antibodies and fragments thereof. It should be noted that the binding proteins of the present invention can also react with (i.e., bind to) forms of a β other than the a β spheromers described herein, and such forms of a β can be present in the brain of patients with amyloidosis, such as alzheimer's disease. These a β forms may or may not be oligomeric or globulomeric. The form of a β to which the binding protein of the invention binds includes any form of a β comprising the globulomer epitope (hereinafter referred to as "m 4D 10") with which the mouse/mouse monoclonal antibody m4D10 reacts. m4D10 and its properties are described in WO 2007/062852, which is incorporated herein by reference. Such a β forms are hereinafter referred to as "targeted a β forms". Further, the invention also provides therapeutic methods therewith for inhibiting the activity of the targeted A β forms, and provides compositions and methods for treating diseases associated with the targeted A β forms, particularly amyloidosis such as Alzheimer's disease.

In one aspect, the invention provides a binding protein comprising: a first amino acid sequence at least 90% identical to

Wherein X ¹² Is I or V, X ²⁴ Is A or V, X ²⁹ Is V or L, X ⁴⁸ Is V or L, X ⁴⁹ Is S or G, X ⁶⁷ Is F or L, X ⁷¹ Is R or K, X ⁷⁶ Is N or S, and X ⁷⁸ Is L or V; or

Wherein X ¹ Is Q or E, X ²⁷ Is a group of G or F,X ²⁹ is I or L, X ³⁷ Is I or V, X ⁴⁸ Is I or L, X ⁶⁷ Is V or L, X ⁷¹ Is V or K, X ⁷⁶ Is N or S, and X ⁷⁸ Is F or V;

and a second amino acid sequence at least 90% identical to:

wherein X ⁷ Is S or T, X ¹⁵ Is L or P, X ⁴¹ Is F or L, X ⁴² Is Q or L, X ⁴⁴ Is R or K, and X ⁵⁰ Is R or Q.

In a further aspect of the invention, the binding protein described above comprises a first amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10 and 11. In a still further aspect of the invention, the binding protein described above comprises a first amino acid sequence selected from the group consisting of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

In another aspect of the invention, a binding protein as described above comprises a second amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: 12, 13, 14, 15 and 16. In a further aspect of the invention, the binding protein described above comprises a second amino acid sequence selected from the group consisting of SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

In one aspect of the invention, a binding protein as described above comprises a first amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10 and 11, and a second amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16. In a further aspect of the invention, the binding protein described above comprises a first amino acid sequence selected from the group consisting of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11; and a second amino acid sequence selected from the group consisting of SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

In a particular aspect of the invention, the binding protein described above comprises a first amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO 6; and a second amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 14. In a further specific aspect of the invention, the binding protein described above comprises a first amino acid sequence as shown in SEQ ID NO. 6 and a second amino acid sequence as shown in SEQ ID NO. 14.

In a particular aspect of the invention, the binding protein described above comprises a first amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID No. 10; and a second amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO. 14. In a further specific aspect of the invention, the binding protein described above comprises a first amino acid sequence shown in SEQ ID NO. 10 and a second amino acid sequence shown in SEQ ID NO. 14.

In one aspect, a binding protein described herein is an antibody. Such antibodies can be, for example, immunoglobulin molecules, disulfide-bonded Fv, monoclonal antibodies (mabs), single chain Fv (scFv), chimeric antibodies, single domain antibodies, CDR-grafted antibodies, diabodies, humanized antibodies, multispecific antibodies, Fab, dual specific antibodies, Dual Variable Domain (DVD) junctionsHybrid molecules, Fab ', bispecific antibodies, F (ab') ₂ Or Fv.

When the binding protein described herein is an antibody, it comprises at least one variable heavy chain corresponding to a first amino acid sequence as defined above, and at least one variable light chain corresponding to a second amino acid sequence as defined above. For example, an antibody of the invention comprises (i) at least one variable heavy chain comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, and (ii) at least one variable light chain comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, 14, 15 and 16. In a particular aspect of the invention, an antibody of the invention comprises (i) at least one variable heavy chain comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO 6 or SEQ ID NO 10, and (ii) at least one variable light chain comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO 14.

The binding proteins described herein may further comprise (in addition to the first and second amino acid sequences) another moiety, which may be another amino acid sequence or other chemical moiety. For example, an antibody of the invention may comprise a heavy chain immunoglobulin constant domain. The heavy chain immunoglobulin constant domain may be selected from the group consisting of a human IgM constant domain, a human IgG4 constant domain, a human IgG1 constant domain, a human IgE constant domain, a human IgG2 constant domain, a human IgG3 constant domain, and a human IgA constant domain. In another aspect, the binding protein of the invention further comprises a heavy chain constant region having an amino acid sequence selected from the group consisting of SEQ ID NO 25 and SEQ ID NO 26, in addition to a light chain constant region having an amino acid sequence selected from the group consisting of SEQ ID NO 27 and SEQ ID NO 28. In a particular aspect of the invention, the binding proteins described herein comprise a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO 6 or SEQ ID NO 10; a variable light chain comprising the amino acid sequence set forth in SEQ ID NO. 14; a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO. 25; and a light chain constant region having the amino acid sequence set forth in SEQ ID NO. 27. In a further specific aspect of the invention, the binding proteins described herein comprise a first amino acid sequence shown in SEQ ID NO 46 or SEQ ID NO 47, and a second first amino acid sequence shown in SEQ ID NO 48.

The binding proteins described herein, e.g., antibodies, may further comprise a therapeutic agent, an imaging agent, a residue capable of promoting the formation of an immunoadhesion molecule and/or another functional molecule (e.g., another peptide or protein). The imaging agent may be a radiolabel including, but not limited to ³ H、 ¹⁴ C、 ³⁵ S、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I、 ¹⁷⁷ Lu、 ¹⁶⁶ Ho and ¹⁵³ sm; an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

The binding proteins of the invention may be glycosylated. According to one aspect of the invention, the glycosylation pattern is a human glycosylation pattern.

In one aspect of the invention, the above-described binding proteins bind to a β forms comprising a globulomer epitope with which the murine monoclonal antibody m4D10 is reactive (i.e., targets the a β form). In particular, the above binding proteins bind to amyloid beta (20-42) globulomers as described herein.

In one aspect of the invention, the binding proteins described herein are capable of modulating the biological function of the A β (20-42) globulomer. In a further aspect of the invention, the binding proteins described herein are capable of neutralizing A β (20-42) globulomer activity.

The binding proteins of the invention may exist as crystals. In one aspect, the crystal is a carrier-free pharmaceutically controlled release crystal. In another aspect, the crystallized binding protein has a longer half-life in vivo than its soluble counterpart. In another aspect, the crystallized binding protein retains biological activity after crystallization.

The invention also provides an isolated nucleic acid encoding any of the binding proteins disclosed herein. Further embodiments provide a vector comprising the nucleic acid. The vector may be selected from pcDNA, pTT (Durocher et al, Nucleic Acids Research 30 (2), 2002), pTT3 (pTT with additional multiple cloning sites), pEFBOS (Mizushima and Nagata, Nucleic Acids Research 18 (17), 1990), pBV, pJV and pBJ.

In another aspect of the invention, a host cell is transformed with a vector as disclosed above. According to one embodiment, the host cell is a prokaryotic cell, including but not limited to E.coli. In related embodiments, the host cell is a eukaryotic cell selected from the group consisting of a protist cell, an animal cell, a plant cell, and a fungal cell. The animal cell may be selected from mammalian cells, avian cells and insect cells. According to one aspect of the invention, the mammalian cells are selected from CHO and COS and the fungal cells are yeast cells such as Saccharomyces cerevisiae (S.cerevisiae)Saccharomyces cerevisiae) And the insect cell is an insect Sf9 cell.

Further, the present invention provides a method of producing a binding protein as disclosed herein, comprising culturing any one of the host cells disclosed herein in a culture medium under conditions and for a time suitable for production of the binding protein. Another embodiment provides a binding protein of the invention produced according to the methods disclosed herein. In another embodiment, the invention provides a binding protein produced according to the method disclosed above.

The invention also provides pharmaceutical compositions comprising a binding protein, e.g., an antibody, as disclosed herein and a pharmaceutically acceptable carrier.

One embodiment of the present invention provides a composition for releasing a binding protein described herein, wherein the composition comprises a formulation, which in turn comprises a crystallized binding protein as disclosed above, e.g., a crystallized antibody and an ingredient; and at least one polymeric carrier. In one aspect, the polymeric carrier is a polymer selected from one or more of the following: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (beta-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide), poly ((organo) phosphazene), poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols), albumin, alginates, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends and copolymers thereof. In another aspect, the ingredient is selected from: albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-beta-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

The invention also relates to a method of inhibiting (i.e. reducing) the activity of an a β (20-42) globulomer (or any other targeted a β form), comprising contacting the targeted a β form with one or more binding proteins of the invention, such that the activity of the targeted a β form is inhibited (i.e. reduced). In a particular embodiment, the activity is inhibited in vitro. Such methods can include adding a binding protein of the invention to a sample or cell culture containing or suspected of containing the targeted a β form, such as a sample derived from the subject (e.g., whole blood, cerebrospinal fluid, serum, tissue, etc.), in order to inhibit (i.e., reduce) the activity of the a β form in the sample. Alternatively, the activity of the targeted a β form may be inhibited (i.e., decreased) in a subject. Thus, the present invention further relates to a binding protein as described herein for use in inhibiting (i.e. reducing) the activity of a targeted a β form in a subject, comprising contacting said a β form with one or more binding proteins of the invention, such that the activity of said a β form is inhibited (i.e. reduced).

In a related aspect, the invention provides methods for inhibiting (i.e., reducing) the activity of a targeted form of a β in a subject suffering from a disease or disorder in which the activity of the form of a β is detrimental. In one embodiment, the method comprises administering to the subject at least one binding protein disclosed herein, such that the activity of the targeted a β form in the subject is inhibited (i.e., reduced). Accordingly, the present invention provides an a β binding protein as described herein for use in inhibiting (i.e. reducing) a targeted a β form in a subject suffering from a disease or disorder as described herein, wherein at least one binding protein as disclosed herein is administered to the subject such that the activity of said a β form in the subject is inhibited (i.e. reduced).

In a related aspect, the invention provides methods for treating (e.g., curing, inhibiting, ameliorating, delaying the onset of, or preventing the recurrence or recurrence of a disease or condition selected from the group consisting of: alpha 1-antitrypsin deficiency, C1-inhibitor deficiency angioedema, antithrombin deficiency thromboembolic disease, Kuru, Creutzfeld-Jacob disease/scrapie, bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker disease, familial fatal insomnia, Huntington's disease, spinocerebellar ataxia, Machado-Joseph atrophy, dentate-pallidopallidoluysian atrophy, frontotemporal dementia, sickle cell anemia, labile hemoglobin inclusion body hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic AL amyloidosis, nodular AL amyloidosis, systemic AA amyloidosis, prostate amyloidosis, hemodialysis amyloidosis, hereditary (iceland) cerebrovascular disease, Huntington's disease, familial visceral amyloidosis, familial polyneuropathy, Crohn's disease, Creutzfeldt-Jakob disease, Crohn's disease, and disease, Familial visceral amyloidosis, senile systemic amyloidosis, familial amyloid neuropathy, familial cardiac amyloidosis, Alzheimer's disease, Down's syndrome, medullary thyroid carcinoma, and type 2 diabetes (T2 DM). In particular embodiments, the disease or disorder is an amyloidosis such as alzheimer's disease or down's syndrome. In one embodiment, the method comprises the step of administering any one of the a β binding proteins disclosed herein, thereby allowing treatment. In another embodiment, the invention provides a method of treating a subject having a disease or disorder selected from those disclosed herein, comprising the step of administering any one of the a β binding proteins disclosed herein simultaneously with or subsequent to the administration of one or more additional therapeutic agents. Accordingly, the present invention provides an a β binding protein disclosed herein for use in treating a subject having a disease or disorder disclosed herein, comprising the step of administering any one of the binding proteins disclosed herein simultaneously with or subsequent to the administration of one or more additional therapeutic agents. For example, the additional therapeutic agent is selected from the therapeutic agents listed herein.

The binding proteins disclosed herein and pharmaceutical compositions comprising the binding proteins are administered to a subject by at least one mode selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavity, intracavitary, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

In another embodiment, the present invention provides a method for detecting a targeted a β form in a sample, comprising (i) contacting said sample with one or more binding proteins of the present invention, and (ii) detecting complex formation between one or more of said binding proteins and an element of said sample, wherein the formation or increased formation of a complex in the sample, relative to a control sample, is indicative of the presence of said a β form in the sample. The sample may be a biological sample (e.g., whole blood, cerebrospinal fluid, serum, tissue, etc.) or a cell culture containing or suspected of containing the a β form obtained from a subject suspected of having a disease or disorder as disclosed herein. Control samples were either free of the a β form or obtained from patients without the disease as described above. The presence of a complex between the binding protein and a sample element obtained from a patient suspected of having alzheimer's disease is indicative of a diagnosis of such a disease in the patient.

In alternative embodiments, detection of the targeted a β form may be performed in vivo, for example, by in vivo imaging in a subject. To this end, one or more binding proteins of the invention may be administered to a subject or a control subject under conditions that allow binding of the protein to a targeted a β form and detecting complex formation between one or more of the binding proteins and the a β form, wherein the formation or increased formation of the complex in the subject relative to the control subject is indicative of the presence of the a β form in the subject. The subject may be a subject known or suspected to have a disorder or disease in which activity of the targeted a β form is detrimental.

Drawings

FIG. 1 illustrates the amino acid sequence of the variable light chain of a humanized 4D10 antibody comprising J κ 2 and V κ A17/2-30 framework regions (SEQ ID NO: 1). All CDR regions are underlined.

FIG. 2 illustrates the amino acid sequence of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 (hJH 6) and VH3_53 framework regions (SEQ ID NO: 2). All CDR regions are underlined.

FIG. 3 illustrates the amino acid sequence of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions (SEQ ID NO: 3). All CDR regions are underlined.

FIG. 4 illustrates the amino acid sequence of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 (hJH 6) and the VH3_53 framework region (SEQ ID NO: 4). All CDR regions are underlined.

FIG. 5 illustrates the amino acid sequence (SEQ ID NO: 5) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH3_53 framework regions with the VH3 consensus variation I12V. All CDR regions are underlined.

FIG. 6 illustrates the amino acid sequence (SEQ ID NO: 6) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH3_53 framework regions with VH3 consensus change I12V and framework back-mutations A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V. All CDR regions are underlined.

FIG. 7 illustrates the amino acid sequence (SEQ ID NO: 7) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH3_53 framework regions with framework back mutations V29L and R71K. All CDR regions are underlined.

FIG. 8 illustrates the amino acid sequence of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions (SEQ ID NO: 8). All CDR regions are underlined.

FIG. 9 illustrates the amino acid sequence (SEQ ID NO: 9) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions with Q1E changes to prevent N-terminal pyroglutamate formation. All CDR regions are underlined.

FIG. 10 illustrates the amino acid sequence (SEQ ID NO: 10) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions with Q1E changes to prevent N-terminal pyroglutamate formation, and framework back mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V. All CDR regions are underlined.

FIG. 11 illustrates the amino acid sequence (SEQ ID NO: 11) of the variable heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions with Q1E changes to prevent N-terminal pyroglutamate formation, and framework back mutations G27F, I29L and V71K. All CDR regions are underlined.

FIG. 12 illustrates the amino acid sequence of the variable light chain of a humanized 4D10 antibody comprising J κ 2 and V κ A17/2-30 framework regions (SEQ ID NO: 12). All CDR regions are underlined.

FIG. 13 illustrates the amino acid sequence (SEQ ID NO: 13) of the variable light chain of humanized 4D10 antibody comprising J κ 2 and V κ A17/2-30 framework regions having V κ 2 consensus changes S7T, L15P, Q37L, R39K and R45Q. All CDR regions are underlined.

FIG. 14 illustrates the amino acid sequence (SEQ ID NO: 14) of the variable light chain of humanized 4D10 antibody comprising J.kappa.2 and V.kappa.A 17/2-30 framework regions having V.kappa.2 consensus changes S7T, L15P, Q37L, R39K and R45Q and framework back mutation F36L. All CDR regions are underlined.

FIG. 15 illustrates the amino acid sequence (SEQ ID NO: 15) of the variable light chain of humanized 4D10 antibody comprising J.kappa.2 and V.kappa.A 17/2-30 framework regions having V.kappa.2 consensus changes S7T and Q37L. All CDR regions are underlined.

FIG. 16 illustrates the amino acid sequence (SEQ ID NO: 16) of the variable light chain of humanized 4D10 antibody comprising J.kappa.2 and V.kappa.A 17/2-30 framework regions having V.kappa.2 consensus changes S7T, Q37L, and R39K. All CDR regions are underlined.

Figure 17 illustrates the amino acid sequence alignment of the variable heavy chains of murine monoclonal antibody 4D10 (m 4D 19) and humanized 4D10 antibody (4D 10 hum) comprising human JH6 (hJH 6) and VH3_53 framework regions. All CDR regions are printed in bold letters. X at position 12 is I or V, X at position 24 is A or V, X at position 29 is V or L, X at position 48 is V or L, X at position 49 is S or G, X at position 67 is F or L, X at position 71 is R or K, X at position 76 is N or S, and X at position 78 is L or V.

Figure 18 illustrates an amino acid sequence alignment of the variable heavy chains of murine monoclonal antibody 4D10 (m 4D 19) and humanized 4D10 antibody (4D 10 hum) comprising human JH6 and VH4_59 framework regions. All CDR regions are printed in bold letters. X at position 1 is Q or E, X at position 27 is G or F, X at position 29 is I or L, X at position 37 is I or V, X at position 48 is I or L, X at position 67 is V or L, X at position 71 is V or K, X at position 76 is N or S, and X at position 78 is F or V.

FIG. 19 illustrates an amino acid sequence alignment of the variable light chains of murine monoclonal antibody 4D10 (m 4D 19) and humanized 4D10 antibody (4D 10 hum) comprising the framework regions of Jkappa 2 and Vkappa A17/2-30. All CDR regions are printed in bold letters. X at position 7 is S or T, X at position 15 is L or P, X at position 41 is F or L, X at position 42 is Q or L, X at position 44 is R or K, and X at position 50 is R or Q.

Fig. 20A and B show platelet factor 4 (PF-4) cross-reactivity of humanized monoclonal antibodies 4D10hum #1 and 4D10hum #2, human/mouse chimeric antibody h1G5 (positive control) and human polyclonal antibody hIgG1 (negative control) in (a) cynomolgus monkey plasma and (B) human plasma as determined by sandwich ELISA. Binding of PF-4 to the immobilized antibody was detected.

Fig. 21A and B show platelet factor 4 (PF-4) cross-reactivity of humanized monoclonal antibodies 4D10hum #1 and 4D10hum #2, human/mouse chimeric antibody h1G5 (positive control) and human polyclonal antibody hIgG1 (negative control) in (a) cynomolgus monkey plasma and (B) human plasma as determined by aligned sandwich ELISA. The antibody was captured on a plate by immobilized anti-mouse IgG. Binding of PF-4 to the captured antibody was detected.

FIGS. 22A and B show platelet factor 4 (PF-4) cross-reactivity of murine monoclonal antibodies m4D10 and m1G5, anti-human PF-4 antibody (positive control), and IgG2A (negative control) in (A) cynomolgus monkey plasma and (B) human plasma as determined by sandwich ELISA. Binding of PF-4 to the immobilized antibody was detected.

FIGS. 23A and B show platelet factor 4 (PF-4) cross-reactivity of murine monoclonal antibodies m4D10 and m1G5, anti-human PF-4 antibody (positive control), and IgG2a (negative control) in (A) cynomolgus monkey plasma and (B) human plasma as determined by an alignment sandwich ELISA. The antibody was captured on a plate by immobilized anti-mouse IgG. Binding of PF-4 to the captured antibody was detected.

FIG. 24 illustrates the amino acid sequence (SEQ ID NO: 46) of the heavy chain of a humanized 4D10 antibody comprising human JH6 and VH3_53 framework regions with VH3 consensus change I12V and framework back mutations A24V, V29L, V48L, S49G, F67L, R71K, N76S, and L78V; and an Ig γ -1 constant region. All CDR regions are underlined.

FIG. 25 illustrates the amino acid sequence (SEQ ID NO: 47) of the heavy chain of a humanized 4D10 antibody comprising human JH6 and VH4_59 framework regions with Q1E changes to prevent N-terminal pyroglutamate formation, and framework back mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V; and an Ig γ -1 constant region. All CDR regions are underlined.

FIG. 26 illustrates the amino acid sequence (SEQ ID NO: 48) of the light chain of humanized 4D10 antibody comprising J κ 2 and V κ A17/2-30 framework regions with V κ 2 consensus changes S7T, L15P, Q37L, R39K and R45Q and the framework back mutation F36L; and an Ig kappa constant region. All CDR regions are underlined.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in any case potentially ambiguous, the definition provided herein preferably overrides any dictionary or external definition. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms is not limiting. In addition, terms such as "element" or "component" include elements and components that comprise one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly employed in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly implemented in the art or as described herein. Nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and laboratory procedures and techniques thereof, are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

The present invention relates to a β binding proteins, in particular anti a β antibodies or a β binding portions thereof, in particular those that bind to a β (20-42) globulomers. These a β binding proteins are capable of distinguishing not only other forms of a β peptide, in particular monomeric and fibrillar (fibrils), but also non-truncated forms of a β globulomer. Accordingly, the present invention relates to A β binding proteins having a binding affinity for the A β (20-42) globulomer that is greater than the binding affinity of such A β binding proteins to the A β (1-42) globulomer.

The term "a β (X-Y)" as used herein refers to an amino acid sequence from amino acid position X to amino acid position Y of the human amyloid β (a β) protein, including X and Y, in particular to an amino acid sequence from amino acid position X to amino acid position Y of amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (SEQ ID NO: 29) (corresponding to amino acid positions 1-43) or any naturally occurring variant thereof, in particular those having at least one mutation selected from the group consisting of: A2T, H6R, D7N, a21G ("formosan"), E22G ("north pole"), E22Q ("netherlands"), E22K ("italy"), D23N ("iowa"), a42T, and a42V, wherein the numbering is relative to the start of the a β peptide, including position X and position Y or sequences with up to three additional amino acid substitutions, none of which can prevent globulomer formation. According to one aspect, there are no additional amino acid substitutions in the portion from amino acid 12 or X (whichever number is higher) to amino acid 42 or Y (whichever number is lower). According to another aspect, there are no additional amino acid substitutions in the portion from amino acid 20 or X (whichever number is higher) to amino acid 42 or Y (whichever number is lower). According to another aspect, there are no additional amino acid substitutions in the portion from amino acid 20 or X (whichever number is higher) to amino acid 40 or Y (whichever number is lower). An "additional" amino acid substitution is herein any deviation from the canonical sequence that is not found in nature.

More specifically, the term "A.beta. (1-42)" as used herein refers to an amino acid sequence from amino acid position 1 to amino acid position 42 of the human A.beta.protein, including 1 and 42, particularly to amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 30) or any naturally occurring variant thereof, particularly those having at least one mutation selected from the group consisting of: A2T, H6R, D7N, a21G ("formosan"), E22G ("north pole"), E22Q ("netherlands"), E22K ("italy"), D23N ("iowa"), a42T, and a42V, wherein the numbering is relative to the start of the a β peptide, including 1 and 42 or sequences with up to three additional amino acid substitutions, none of which can prevent globulomer formation. According to one aspect, there are no additional amino acid substitutions in the portion from amino acid 20 to amino acid 42. Likewise, the term "A.beta. (1-40)" as used herein refers to an amino acid sequence from amino acid position 1 to amino acid position 40 of the human A.beta.protein, including 1 and 40, particularly to amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV (SEQ ID NO: 31) or any naturally occurring variant thereof, particularly those having at least one mutation selected from the group consisting of: A2T, H6R, D7N, a21G ("formosan"), E22G ("north pole"), E22Q ("netherlands"), E22K ("italy"), and D23N ("iowa"), wherein the numbering includes 1 and 40 or sequences with up to three additional amino acid substitutions relative to the start of the a β peptide, none of which can prevent globulomer formation. According to one aspect, there are no additional amino acid substitutions in the portion from amino acid 20 to amino acid 40.

More specifically, the term "A.beta. (12-42)" as used herein refers to an amino acid sequence from amino acid position 12 to amino acid position 42 of the human A.beta.protein, including 12 and 42, particularly to amino acid sequence VHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 32) or any naturally occurring variant thereof, particularly those having at least one mutation selected from the group consisting of: a212G ("formon"), E22G ("north pole"), E22Q ("netherlands"), E22K ("italy"), D23N ("iowa"), a42T, and a42V, wherein the numbering includes 12 and 42 or sequences with up to three additional amino acid substitutions relative to the start of the a β peptide, none of which can prevent globulomer formation. According to one aspect, there are no additional amino acid substitutions in the portion from amino acid 20 to amino acid 42. Likewise, the term "A β (20-42)" as used herein refers to an amino acid sequence from amino acid position 20 to amino acid position 42 of the human amyloid β protein, including 20 and 42, particularly to amino acid sequence F AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 33) or any naturally occurring variant thereof, particularly those having at least one mutation selected from the group consisting of: a21G ("formosan"), E22G ("north pole"), E22Q ("netherlands"), E22K ("italy"), D23N ("iowa"), a42T, and a42V, wherein the numbering includes 20 and 42 or sequences with up to three additional amino acid substitutions relative to the start of the a β peptide, none of which can prevent globulomer formation. According to one aspect, there are no additional amino acid substitutions.

The term "a β (X-Y) globulomer" (a β (X-Y) globulomer) as used herein refers to a soluble, globular, non-covalent association of a β (X-Y) peptides as defined above, with homogeneity and unique physical characteristics. According to one aspect, the A β (X-Y) globulomer is a stable, non-fibrillar, oligomeric assembly of A β (X-Y) peptides, obtainable by incubation with an anionic detergent. In contrast to monomers and fibrils, these globulomers are characterized by a defined assembly number of subunits (e.g., early assembly forms with 4-6 subunits, "oligo A", and late assembly forms with 12-14 subunits, "oligo B", as described in WO 2004/067561). Globulomers have a three-dimensional spherical structure ("fused spheres", see Barghorn et al, J neurohem 95: 834-847, 2005). They may be further characterized by one or more of the following features:

-the N-terminal amino acid X-23, cleavages with promiscuous protease (e.g.thermolysin or the intracellular protease GluC) to obtain a truncated form of the globulomer;

-C-terminal amino acids 24-Y are not accessible to promiscuous proteases and antibodies;

-these globulomers in truncated form maintain the three-dimensional core structure of said globulomers, the core epitope a β (20-Y) having better access in its globulomer configuration.

According to the present invention and in particular for the purpose of assessing the binding affinity of the a β binding proteins of the present invention, the term "a β (X-Y) globulomer" herein especially refers to a product obtainable by a process as described in WO2004/067561, which is incorporated herein by reference. The process comprises unfolding a natural, recombinant or synthetic a β (X-Y) peptide or a derivative thereof; exposing the at least partially unfolded a β (X-Y) peptide or derivative thereof to a detergent, reducing the detergency and continuing the incubation.

For the purpose of unfolding the peptide, hydrogen bond disrupting agents such as Hexafluoroisopropanol (HFIP) may be allowed to act on the protein. When the temperature of action is about 20-50 c, and in particular about 35-40 c, an action time of a few minutes, for example about 10-60 minutes, is sufficient. Subsequent dissolution of the residue evaporated to dryness, for example in concentrated form, in a suitable organic solvent miscible with aqueous buffers such as dimethyl sulfoxide (DMSO), results in a suspension of at least partially unfolded peptide or derivative thereof, which can then be used. If desired, the stock suspension may be stored at a low temperature, for example at about 20℃, for an intermediate period. Alternatively, the peptide or derivative thereof may be absorbed in a slightly acidic, e.g., aqueous solution, e.g., about 10 mM aqueous HCl. After an incubation time of typically a few minutes, insoluble components are removed by centrifugation. At 10,000 gSeveral minutes are advantageous. These method steps can be carried out at room temperature, i.e. at 20-30 ℃. The supernatant obtained after centrifugation contains the a β (X-Y) peptide or derivative thereof and can be stored at low temperature, e.g. at about-20 ℃, for an intermediate period. Subsequent exposure to detergents involves oligomerization of the peptide or derivative thereof to give an intermediate type of oligomer (referred to as oligomer a in WO 2004/067561). For this purpose, letThe detergent is allowed to act on the at least partially unfolded peptide or derivative thereof until sufficient intermediate oligomers have been produced. The use of ionic detergents, especially anionic detergents, is preferred.

According to a particular embodiment, detergents of formula (I) below are used:

R-X，

wherein the radical R is an unbranched or branched alkyl radical having 6 to 20, for example 10 to 14, carbon atoms or an unbranched or branched alkenyl radical having 6 to 20, for example 10 to 14, carbon atoms and the radical X is an acidic radical or a salt thereof, wherein X is selected, for example, from the group-COO-M ⁺ 、-SO ₃ -M ⁺ Etc. and especially-OSO ₃ -M ⁺ And M is ⁺ Is a hydrogen cation or an inorganic or organic cation selected, for example, from the alkali and alkaline earth metal cations and ammonium cations. Of advantage are detergents of the formula (I) in which R is an unbranched alkyl radical, in which particular mention must be made of alk-1-yl radicals. For example, Sodium Dodecyl Sulfate (SDS), lauric acid, the detergent lauryl creatine sodium salt (also known as sarcosyl NL-30 or Gardol @) and oleic acid may be used advantageously. The time of decontamination depends inter alia on whether (and if so, to what extent) the peptide or derivative thereof undergoing oligomerization has unfolded. If, according to the unfolding step, the peptide or derivative thereof has been previously treated with a hydrogen bond disrupting agent, i.e. in particular with hexafluoroisopropanol, an action time in the range of several hours, advantageously about 1 to 20 and in particular about 2 to 10 hours, is sufficient when the action temperature is about 20 to 50 ℃ and in particular about 35 to 40 ℃. If a less unfolded or substantially unfolded peptide or derivative thereof is the starting point, a correspondingly longer action time is advantageous. If the peptide or derivative thereof has been pretreated, for example according to the procedure described above, as an alternative to the treatment with HFIP, or the peptide or derivative thereof is directly oligomerized, a duration of action in the range of about 5-30 hours, and in particular about 10-20 hours, is sufficient when the temperature of action is about 20-50 ℃, and in particular about 35-40 ℃. After incubation, insoluble components are advantageously removed by centrifugation. At 10,000 gSeveral minutes are advantageous. To be selectedThe concentration of detergent in (a) depends on the detergent used. If SDS is used, a concentration in the range of 0.01-1% by weight, for example 0.05-0.5% by weight, for example about 0.2% by weight, proves advantageous. If lauric acid or oleic acid is used, a slightly higher concentration is advantageous, for example in the range of 0.05-2% by weight, for example 0.1-0.5% by weight, for example about 0.5% by weight. The decontamination should occur at a salt concentration approximately in the physiological range. Thus, particularly in the range of 50-500 mM, for example 100-200 mM or at a NaCl concentration of about 140 mM, is advantageous. Subsequent reduction of decontamination and continuation of incubation involved further oligomerisation to give the A β (X-Y) globulomer of the invention (referred to as globulomer B in WO 2004/067561). Since the compositions obtained from the previous step usually contain detergents and salt concentrations in the physiological range, it is advantageous to subsequently reduce the detergency as well as the salt concentration. This may be performed by reducing the concentration of detergent and salt, for example by conveniently diluting with water or a buffer of lower salt concentration, for example Tris-HCl, pH 7.3. Dilution factors in the range of about 2-10, effectively in the range of about 3-8 and especially about 4 have proven suitable. Reduction in detergency can also be achieved by the addition of substances which can neutralise the detergency. Examples of these include substances which are capable of complexing detergents, such as substances which are capable of stabilizing the cells during the purification and extraction measures, for example in particular EO/PO block copolymers, in particular under the trade name Pluronic F68. Similarly alkylated and especially ethoxylated alkylphenols such as Triton X series of ethoxylated t-octylphenol, especially Triton X100, 3- (3-cholamidopropyl dimethylamino) -1-propane sulfonate (CHAPS) or alkoxylated and especially ethoxylated sorbitan fatty esters such as those of the Tween series, especially Tween 20, in a range of concentrations about or above a particular critical micelle concentration may be used. Subsequently, the solution is incubated until sufficient A β (X-Y) globulomers of the invention have been produced. When the temperature of action is about 20-50 ℃ and in particular about 35-40 ℃, in the range of several hours, for example in the range of about 10-30 hours or in the range of about 15- An action time in the range of 25 hours is sufficient. The solution may then be concentrated and possible debris may be removed by centrifugation. Here again, at 10,000gSeveral minutes proved to be advantageous. The supernatant obtained after centrifugation contains the A β (X-Y) globulomers of the invention. The A β (X-Y) globulomers of the invention can finally be recovered in a manner known per se, for example by ultrafiltration, dialysis, precipitation or centrifugation. For example, electrophoretic separation of A β 0 (X-Y) globulomers under denaturing conditions, e.g., by SDS-PAGE, may produce a double band (e.g., having an apparent molecular weight of 38/48 kDa for A β 1 (1-42)), and these two bands may be combined into one after glutaraldehyde treatment of the globulomers prior to separation. Size exclusion chromatography of globulomers can result in a single peak (e.g., corresponding to a molecular weight of about 100 kDa for A β (1-42) globulomers or about 60 kDa for glutaraldehyde-crosslinked A β (1-42) globulomers), respectively. Starting from the A beta (1-42) peptide, the A beta (12-42) peptide and the A beta (20-42) peptide, the process is particularly suitable for obtaining A beta (1-42) globulomers, A beta (12-42) globulomers and A beta (20-42) globulomers.

In a particular embodiment of the invention, a β (X-Y) globulomer in which X is selected from the number 2.. 24 and Y is as defined above are those which can be achieved by truncating the a β (1-Y) globulomer to a shorter form, wherein X is selected from the number 2.. 24, for example X is 20 or 12, and Y is as defined above, which can be achieved by treatment with a suitable protease. For example, A β (20-42) globulomer may be obtained by performing thermolysin proteolysis on A β (1-42) globulomer, and A β (12-42) globulomer may be obtained by performing intracellular protease GluC proteolysis on A β (1-42) globulomer. When the desired degree of proteolysis is reached, the protease is inactivated in a generally known manner. The resulting globulomer may then be isolated according to the procedures already described herein and, if desired, further processed by further work-up and purification steps. A detailed description of the process is disclosed in WO 2004/067561 which is incorporated herein by reference.

For the purposes of the present invention, A β (1-42) globulomers are in particular those described in example 1a below; the A β (20-42) globulomer is in particular an A β (20-42) globulomer as described in example 1b below, and the A β (12-42) globulomer is in particular an A β (12-42) globulomer as described in example 1c below. According to one aspect of the invention, the globulomer exhibits affinity for neuronal cells and/or exhibits neuromodulatory effects.

According to another aspect of the invention, the globulomer consists of 11-16, e.g. 12-14, A β (X-Y) peptides. According to another aspect of the invention, the term "a β (X-Y) globulomer" refers herein to a globulomer consisting essentially of a β (X-Y) subunits, wherein for example at least 11 of the average 12 subunits are of the a β (X-Y) type, or less than 10% of the globulomers comprise any non-a β (X-Y) peptide, or the content of non-a β (X-Y) peptides is below the detection threshold. More specifically, the term "a β (1-42) globulomer" refers herein to a globulomer consisting essentially of a β (1-42) units as defined above; the term "a β (12-42) globulomer" refers herein to a globulomer consisting essentially of a β (12-42) units as defined above; and the term "A β (20-42) globulomer" refers herein to a globulomer consisting essentially of A β (20-42) units as defined above.

The term "crosslinked a β (X-Y) globulomer" refers herein to a molecule obtainable from an a β (X-Y) globulomer as described above by crosslinking of the constituent units of the globulomer, e.g., chemical crosslinking, aldehyde crosslinking, glutaraldehyde crosslinking. In another aspect of the invention, a cross-linked globulomer is essentially a globulomer in which the units are at least partially held together by covalent bonds rather than by mere non-covalent interactions. For the purposes of the present invention, crosslinked A β (1-42) globulomers are in particular crosslinked A β (1-42) globulomers as described in example 1d below.

The term "Abeta (X-Y) globulomer derivative" as used herein refers in particular to a globulomer labeled by covalent attachment to a group facilitating detection, e.g.a fluorophore such as fluorescein isothiocyanate, phycoerythrin, Aequorea victoria (Aequorea victoria) fluorescent protein, a Zoarcina(Dictyosoma) fluorescent protein, or any combination or fluorescently active derivative thereof; a chromophore; chemiluminescent bodies such as luciferases, especially fireflies of North America (Photinus pyralis) Luciferase, Vibrio fischeri: (Vibrio fischeri) Luciferase, or any combination or chemiluminescent-active derivative thereof; an enzymatically active group such as a peroxidase, e.g., horseradish peroxidase, or any enzymatically active derivative thereof; electron-dense groups such as heavy metal-containing groups, e.g., gold-containing groups; haptens, such as phenol-derived haptens; strong antigenic structures, such as peptide sequences predicted to be antigenic, e.g., by algorithms of Kolaskar and Tongaonkar; an aptamer to another molecule; chelating groups such as hexahistidyl; natural or naturally derived protein structures that mediate further specific protein-protein interactions, such as members of the fos/jun pair; magnetic groups, such as magnetic iron groups; or a radioactive group such as a group comprising 1H, 14C, 32P, 35S, or 125I, or any combination thereof; or a globulomer labeled by covalent or non-covalent high affinity interactions linked to groups that promote inactivation, sequestration, degradation and/or precipitation, such as labeling with groups that promote degradation in vivo, such as ubiquitin, and oligomers of such labels assembled, for example, in vivo; or a globulomer modified by any combination of the above. Such labels and marker groups and methods for attaching them to proteins are known in the art. Labeling and/or tagging may be performed before, during, or after globulomerization. In another aspect of the invention, a globulomer derivative is a molecule obtainable from a globulomer by a labeling and/or labeling reaction. Accordingly, the term "a β (X-Y) monomer derivative" herein especially refers to a β monomers that are labeled or tagged as described for globulomers.

In a further aspect of the invention, the binding proteins described herein bind with high affinity to the A β (20-42) globulomer, e.g., having up to about 10 ^-6 M; up to about 10 ^-7 M; up to about 10 ^-8 M; up to about 10 ^-9 M; up to about 10 ^-10 M; up to about 10 ^-11 M; up to about 10 ^-12 M; and at most 10 ^-13 Dissociation constant (K) of M _D ). In one aspect, the binding proteins described herein bind to the a β (20-42) globulomer with a rate constant (k) as measured by surface plasmon _on ) Selected from: at least about 10 ² M ^-1 s ^-1 (ii) a At least about 10 ³ M ^-1 s ^-1 (ii) a At least about 10 ⁴ M ^-1 s ^-1 (ii) a At least about 10 ⁵ M ^-1 s ^-1 (ii) a And at least about 10 ⁶ M ^-1 s ^-1 . In another aspect, the binding protein has an off-rate constant (k) from the a β (20-42) globulomer selected from the group consisting of _off ): up to about 10 ^-3 s ^-1 (ii) a Up to about 10 ^-4 s ^-1 (ii) a Up to about 10 ^-5 s ^-1 (ii) a And up to about 10 ^-6 s ^-1 . In a particular aspect of the invention, the binding proteins described herein are expressed as 1 × 10 ^-9 - 1× 10 ^-10 The dissociation constant of M binds to the A β (20-42) globulomer. In a further particular aspect of the invention, the binding protein described herein binds to the A β (20-42) globulomer with a rate constant (k) _on ) Is 1X 10 ⁵ - 1× 10 ⁶ M ^-1 s ^-1 . In a further particular aspect of the invention, the binding proteins described herein have a size of 8 × 10 ^-5 - 8× 10 ^-4 s ^-1 With A beta (20-42) globulomer _off ）。

In another aspect of the invention, the binding proteins described herein have a greater binding affinity for the A β (20-42) globulomer than for the A β (1-42) globulomer.

The term "greater affinity" refers herein to the degree of interaction when the equilibrium between unbound a β binding protein and unbound a β globulomer on the one hand and a β binding protein-globulomer complex on the other hand further supports a β binding protein-globulomer complex. Likewise, the term "lower affinity" refers herein to the balance between unbound a β binding protein and unbound a β globulomer on the one hand and a β binding protein-globulomer complex on the other hand further supporting the degree of interaction between unbound a β binding protein and unbound a β globulomer. The term "greater affinity" is synonymous with the term "higher affinity" and the term "lesser affinity" is synonymous with the term "lower affinity".

In related aspects of the invention, the binding proteins described herein bind to the a β (20-42) globulomer with at least 2 times (e.g., at least 3 or at least 5 times), at least 10 times (e.g., at least 20 times, at least 30 times, or at least 50 times), at least 100 times (e.g., at least 200 times, at least 300 times, or at least 500 times), and at least 1,000 times (e.g., at least 2,000 times, at least 3,000 times, or at least 5000 times), at least 10,000 times (e.g., at least 20,000 times, at least 30,000 times, or at least 50,000 times), or at least 100,000 times greater than the binding affinity of the binding protein to the a β (1-42) globulomer.

In a still further aspect of the invention, the binding proteins described herein bind to the A β (1-42) globulomer with relatively high affinity, e.g., having up to about 10 ^-6 M; up to about 10 ^-7 M; up to about 10 ^-8 M; up to about 10 ^-9 M; up to about 10 ^-10 M; up to about 10 ^-11 M; up to about 10 ^-12 M; and at most 10 ^-13 Dissociation constant (K) of M _D ). In one aspect, the binding protein described herein binds to the a β (12-42) globulomer with a rate constant (k) as measured by surface plasmon _on ) Selected from: at least about 10 ² M ^-1 s ^-1 (ii) a At least about 10 ³ M ^-1 s ^-1 (ii) a At least about 10 ⁴ M ^-1 s ^-1 (ii) a At least about 10 ⁵ M ^-1 s ^-1 (ii) a And at least about 10 ⁶ M ^-1 s ^-1 . In another aspect, the binding protein has an off-rate constant (k) from the a β (12-42) globulomer selected from the group consisting of _off ): up to about 10 ^-3 s ^-1 (ii) a Up to about 10 ^-4 s ^-1 (ii) a Up to about 10 ^-5 s ^-1 (ii) a And up to about 10 ^-6 s ^-1 。

In a related aspect of the invention, the binding proteins described herein bind to the A β (20-42) globulomer with a binding affinity that is about 1.1-3 times greater than the binding affinity of the binding protein to the A β (1-42) globulomer.

According to one aspect, the a β binding proteins of the invention bind to at least one a β globulomer as defined above and have a relatively small affinity for at least one non-globulomer form of a β. A β binding proteins of the invention that have a lower affinity for at least one non-globulomer form of A β than for at least one A β globulomer include A β binding proteins that have a greater binding affinity for the A β (20-42) globulomer than to the A β (1-42) monomer. According to an alternative or additional aspect of the invention, the binding affinity of the A β binding protein to the A β (20-42) globulomer is greater than the affinity to the A β (1-40) monomer. In particular, the affinity of A β binding proteins for A β (20-42) globulomers is greater than their affinity for A β (1-40) and A β (1-42) monomers.

The term "a β (X-Y) monomer" as used herein refers to an isolated form of a β (X-Y) peptide, in particular a β (X-Y) peptide form that does not substantially participate in non-covalent interactions with other a β peptides. In practice, the A β (X-Y) monomer is usually provided in the form of an aqueous solution. In a particular embodiment of the invention, the aqueous monomer solution contains 0.05% to 0.2%, for example about 0.1% NH ₄ And (5) OH. In another particular embodiment of the invention, the aqueous monomer solution contains 0.05% to 0.2%, for example about 0.1% NaOH. When used (e.g. for determining the binding affinity of a β binding proteins of the invention), it may be advantageous to dilute the solution in a suitable manner. Further, it is often advantageous to use the solution within 2 hours, in particular within 1 hour and especially within 30 minutes after its preparation.

More specifically, the term "a β (1-40) monomer" refers herein to a β (1-40) monomer formulation as described herein, and the term "a β (1-42) monomer" refers herein to an a β (1-42) formulation as described herein.

Advantageously, the A.beta.binding proteins of the invention bind one or both proteins with low affinityCombinations of monomers, e.g. having 1x10 ^-8 K of M _D Or less affinity, e.g. with 3X10 ^-8 K of M _D Or less, having an affinity of 1x10 ^-7 K of M _D Or less affinity, e.g. 3x10 ^-7 K of M _D Or less affinity, or 1x10 ^-6 K of M _D Or less affinity, e.g. 3x10 ^-5 K of M _D Or less affinity, or 1X10 ^-5 K of M _D Or less affinity.

According to one aspect of the invention, the binding affinity of the a β binding proteins of the invention to the a β (20-42) globulomer is at least 2 times, such as at least 3 times or at least 5 times, at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, at least 100 times, such as at least 200 times, at least 300 times or at least 500 times, at least 1,000 times, such as at least 2,000 times, at least 3,000 times or at least 5,000 times, at least 10,000 times, such as at least 20,000 times, at least 30,000 or at least 50,000 times, or at least 100,000 times higher than the binding affinity of the a β binding protein to one or both monomers.

A β binding proteins of the invention that have a lower affinity for at least one non-globulomeric form of A β than for at least one A β globulomer further include A β binding proteins that have a greater binding affinity for A β (20-42) globulomers than for A β (1-42) fibrils. According to an alternative or additional aspect of the invention, the binding affinity of the A β binding protein to the A β (20-42) globulomer is greater than the affinity to the A β (1-40) filament. According to a particular embodiment, the invention relates to a β binding proteins having a binding affinity to the a β (20-42) globulomer which is greater than its binding affinity to the a β (1-40) and a β (1-42) fibrils.

The term "fibril" refers herein to an assembled molecular structure comprising non-covalently bound individual a β (X-Y) peptides, which exhibits a fibril structure in an electron microscope, which binds congo red and subsequently exhibits birefringence under polarized light, and whose X-ray diffraction pattern is a cruciform β structure. In another aspect of the invention, the fibrils are molecules obtainable by such a processStructure, said process comprising self-induced polymeric aggregation of a suitable a β peptide in the absence of detergent, e.g. in 0.1M HCl, resulting in aggregate formation of more than 24 or more than 100 units. This process is well known in the art. Advantageously, the Α β (X-Y) fibrils are used in the form of an aqueous solution. In a particular embodiment of the invention, the aqueous filament solution is prepared by: dissolving A beta peptide in 0.1% NH ₄ In OH, it was treated with 20 mM NaH ₂ PO ₄ 140 mM NaCl, pH 7.4, diluted 1:4, followed by readjustment of the pH to 7.4, and incubation of the solution at 37 ℃ for 20 hours, followed by 10,000gCentrifuged for 10 min and resuspended in 20 mM NaH ₂ PO ₄ 140 mM NaCl, pH 7.4. The term "Α β (X-Y) filament" herein also refers to a filament comprising Α β (X-Y) subunits, wherein for example on average at least 90% of the subunits have Α β (X-Y) type, at least 98% of the subunits have Α β (X-Y) type, or the content of non- Α β (X-Y) peptide is below the detection threshold. More specifically, the term "a β (1-42) filament" refers herein to a β (1-42) filament formulation as described in example 3.

Advantageously, the a β binding proteins of the invention bind to one or both of the fibrils with low affinity, e.g. with 1x10 ^-8 K of M _D Or less affinity, e.g. with 3X10 ^-8 K of M _D Or less affinity, 1X10 ^-7 K of M _D Or less affinity, e.g. 3x10 ^-7 K of M _D Or less affinity, or 1x10 ^-6 K of M _D Or less affinity, e.g. 3x10 ^-5 K of M _D Or less affinity, or 1x10 ^-5 K of M _D Or less affinity.

According to one aspect of the invention, the binding affinity of an a β binding protein of the invention to an a β (20-42) globulomer is at least 2 fold, such as at least 3 fold or at least 5 fold, at least 10 fold, such as at least 20 fold, at least 30 fold or at least 50 fold, at least 100 fold, such as at least 200 fold, at least 300 fold or at least 500 fold, at least 1,000 fold, such as at least 2,000 fold, at least 3,000 fold or at least 5,000 fold, at least 10,000 fold, such as at least 20,000 fold, at least 30,000 or at least 50,000 fold, or at least 100,000 fold higher than the binding affinity of an a β binding protein to one or both of the fibrils.

According to a particular embodiment, the present invention relates to a β binding proteins having a relatively small affinity for at least one a β globulomer, in particular a β (20-42) globulomer, for a β in monomeric and fibrillar form. These a β binding proteins are sometimes referred to as globulomer-specific a β binding proteins.

Binding proteins of the invention such as humanized antibody 4D10 (4D 10 hum) include globulomer-specific binding proteins that predominantly recognize the form of a β (20-42) globulomer rather than a β (1-40) monomer, a β (1-42) monomer, a β fibril, or a standard preparation of sAPP (i.e., insoluble a β precursor) as compared to, for example, competing antibodies such as m266 and 3D 6. Such specificity for globulomers is important because a β specifically targeting the globulomer form with humanized 4D10 will: 1) avoiding targeting of insoluble amyloid deposits, the binding to which would explain the inflammatory side effects observed during immunization with insoluble a β; 2) excess a β monomers and APP with pre-recognition physiological functions are reported (Plan et al, J Neurosci 23: 5531-5535, 2003; and 3) increasing the bioavailability of the antibody as it will be unmasked or inaccessible by extensive binding to insoluble precipitates.

PF-4 is a small, 70 amino acid cytokine belonging to the CXC chemokine family, and is also known as chemokine (C-X-C motif) ligand 4 (CXCL 4). PF-4 is released from platelet-activating alpha-granules during platelet aggregation and promotes blood coagulation by modulating the effects of heparin-like molecules. Because of these functions, it is predicted to be involved in wound repair and inflammation (Eismann et al, Blood 76 (2): 336-44, 1990). PF-4 is commonly found in complexes with proteoglycans and can form complexes with the anticoagulant heparin, which is used as a pharmacological treatment for thrombosis. It has well-described pathological functions in heparin-induced thrombocytopenia (HIT), a specific autoimmune response to anticoagulant heparin administration (Warkentin, n. engl. j. med. 356 (9): 891-3, 2007), where heparin: the PF4 complex is an antigen. PF4 autoantibodies have also been found in patients with thrombosis and similar characteristics to prior administrations of HIT but without heparin (Warkentin et al, Am. j. med. 121 (7): 632-6, 2008). Heparin-induced thrombocytopenia is characterized by the development of thrombocytopenia (low platelet count), and in addition HIT is prone to thrombosis. Given these functions and implications of PF-4 in pathological processes, it can be concluded that administration of a binding protein (e.g., an antibody) that shows binding (e.g., cross-reactivity) with PF-4 present in a subject can affect the PF-4 function and thereby lead to adverse (side) effects. The extent and nature of such adverse effects may vary depending on parameters such as the location and size of the epitope on PF-4, the binding strength and nature of the respective binding protein.

According to one aspect of the invention, the binding protein of the invention shows no or low binding to platelet factor 4 (PF-4). The cross-reactivity with PF-4 can be assessed by using standardized in vitro immunoassays such as ELISA, dot blot or BIAcore analysis.

According to a particular embodiment, the cross-reactivity of a binding protein as defined herein with PF-4 refers to the ratio of the values obtained for said binding protein and a reference anti-PF-4 antibody by: (i) performing a sandwich ELISA with a: -1: 3 dilution series of human or cynomolgus monkey plasma of about 1:3.16 to about 1:3160 (final plasma dilution) (e.g., as described in examples 3.1 and 3.2), (ii) plotting the detected signal (y-axis) against log-transformed plasma dilutions (x-axis), and (iii) determining the area under the curve (AUC, or total peak area) from these non-curve-fitted data over the measurement range (final plasma dilutions of about 1:3.16 to about 1: 3160). According to a particular embodiment of the invention, the cross-reaction with PF-4 as determined by sandwich ELISA comprises the following: a specific amount of binding protein under investigation or a reference anti-PF-4 antibody or conveniently a suitable dilution thereof, e.g. 100 μ l of 10 μ g/ml binding protein or antibody solution in 100 mM sodium bicarbonate pH 9.6, is used for protein uptake into coated wells of a microtiter plate; the plate was then washed, blocked and washed again; followed by serial contact with about 1:3.16 to about 1:3160 (final plasma dilution) of cynomolgus monkey or human plasma, e.g., human plasma spiked with human PF-4, followed by detection of PF-4 bound to each well, e.g., via PF-4 specific primary antibodies, enzyme conjugated secondary antibodies, and colorimetric reactions.

As used herein, a "reference anti-PF-4 antibody" is an antibody, particularly a monoclonal antibody, that specifically reacts with PF-4, particularly human (HPF 4). Such antibodies can be obtained by: an antigen comprising human PF-4, e.g., human PF-4 having amino acid sequence EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO: 70), is provided, an antibody reservoir is exposed to the antigen and an antibody that specifically binds to human PF-4 is selected from the antigen reservoir. The antibody may optionally be affinity purified using an immunogen (human PF-4). Such reference anti-PF 4 antibodies are commercially available, e.g., monoclonal anti-HPF 4 antibody, Abcam cat No.: ab 49735.

According to another particular embodiment, the cross-reactivity of a binding protein as defined herein with PF-4 refers to the ratio of AUC values obtained for said binding protein and a reference anti-PF-4 antibody by: (i) performing an aligned sandwich ELISA with human or cynomolgus monkey plasma and a ∼ 1:3 dilution series of about 10 ng/ml to about 10000 ng/ml (final concentration) of binding protein and reference anti-PF-4 antibody (e.g. as described in examples 3.3 and 3.4), (ii) plotting the detected signal (y-axis) against log-transformed binding protein or reference anti-PF-4 antibody concentration (x-axis), and (iii) determining the area under the curve (AUC, or total area of peaks) from these non-curve-fitted data in the measurement range (about 10 ng/ml to about 10000 ng/ml of binding protein or reference antibody concentration). According to a particular embodiment of the invention, the cross-reaction with PF-4 by the alignment sandwich ELISA assay comprises the following: with a specific amount of aligned antibody suitable for capturing the binding protein under study and the reference anti-PF-4 antibody, e.g. 50 μ g/ml Fc specific anti-mouse IgG at 100 μ l/well, Sigma catalog No.: m3534 in 100 mM sodium bicarbonate pH 9.6) coated wells of a protein-absorbing microtiter plate; the plate was then washed, blocked and washed again; followed by contact with a 1:3 dilution series of about 10 ng/ml to about 10000 ng/ml (final concentration) of the binding protein under study or a reference anti-PF-4 antibody; after another washing step, the plates are contacted with e.g. a 1:10 dilution of human or cynomolgus plasma, e.g. human plasma spiked with human PF-4, followed by detection of PF-4 bound to the plates, e.g. by means of a PF-4 specific primary antibody, an enzyme conjugated secondary antibody and a colorimetric reaction.

According to one aspect of the invention, the a β binding protein of the invention has a cross-reactivity with PF-4 that is less than the corresponding cross-reactivity of a reference anti-PF-4 antibody, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold less, when assayed in cynomolgus monkey plasma via sandwich ELISA as described herein; and/or is less than the corresponding cross-reaction of a reference anti-PF-4 antibody, e.g., or at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold less, when assayed via sandwich ELISA as described herein with human plasma.

According to another aspect of the invention, the a β binding protein of the invention has a cross-reactivity with PF-4 that is less than the corresponding cross-reactivity of a reference anti-PF-4 antibody, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 80-fold, or at least 115-fold, when analyzed with cynomolgus monkey plasma via an aligned sandwich ELISA as described herein; and/or is less than the corresponding cross-reaction of a reference anti-PF-4 antibody, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold less, when assayed in human plasma via sandwich ELISA as described herein.

According to another aspect of the invention, the a β binding protein of the invention cross-reacts with PF-4 less than the corresponding cross-reaction of a reference anti-PF-4 antibody, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold less, when assayed in cynomolgus monkey plasma via a sandwich ELISA and an aligned sandwich ELISA as described herein.

According to another aspect of the invention, the a β binding protein of the invention has a cross-reactivity with PF-4 that is less than, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold less than the corresponding cross-reactivity of a reference anti-PF-4 antibody when assayed in human plasma via a sandwich ELISA and an aligned sandwich ELISA as described herein.

According to another aspect of the invention, the a β binding protein of the invention cross-reacts less than, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold less than the corresponding cross-reaction of a reference anti-PF-4 antibody when analyzed with cynomolgus monkey and human plasma via sandwich ELISA and an aligned sandwich ELISA as described herein.

The term "polypeptide" as used herein refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide, and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses natural or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, due to its origin or derivative source, does not bind to components with which it naturally binds in its natural state; substantially free of other proteins from the same species; expressed by cells from different species; or do not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from the components with which it is naturally associated. Proteins may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

As used herein, the term "recovering" refers to the process of rendering a chemical species, such as a polypeptide, substantially free of naturally associated components by separation, for example, using protein purification techniques well known in the art.

As used herein, the term "specifically binds" in reference to an interaction of an antibody, protein or peptide with a second chemical species means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to specific protein structures rather than to general proteins. If the antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in the reaction of labeled "A" with the antibody will reduce the amount of labeled A bound to the antibody.

As used herein, the term "antibody" refers broadly to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof, which retains the essential epitope binding characteristics of an Ig molecule. Such functional fragment, mutant, variant or derivative antibody formats are known in the art. Non-limiting embodiments of which are discussed below. As used herein, a "full-length antibody" refers to an Ig molecule comprising four polypeptide chains (two heavy chains and two light chains). The chains are typically linked to each other via disulfide bonds. Each heavy chain comprises a heavy chain variable region (also referred to herein as a "variable heavy chain" or abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains-CH 1, CH2, and CH 3. Each light chain comprises a light chain variable region (also referred to herein as a "variable light chain" or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a CL domain. The VH and VL regions may be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs), interspersed with more conserved regions known as Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA1, and IgA 2), or subclass.

As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody, an "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a β (20-42) globulomer), i.e., are functional fragments of an antibody. It has been shown that the antigen binding function of an antibody can be performed by one or more fragments of a full-length antibody. Such antibodiesEmbodiments may also be bispecific, bispecific or multispecific, specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (v) dAb fragments comprising a single variable domain (Ward et al, Nature 341: 544-546, 1989; Winter et al, WO 90/05144A 1, incorporated herein by reference); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423-. Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker is used that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and creating two antigen binding sites (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Such antigen binding moieties are known in the art (Kontermann and Dubel editions, Antibody Engineering, Springer-Verlag. New York. 790, p. 2001, ISBN 3-540-41354-5).

As used herein, the term "antibody" also encompasses antibody constructs. The term "antibody construct" as used herein refers to a construct comprising one or more antigen-binding portions of the invention linked to a linker polypeptide or immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

Immunoglobulin constant domains refer to heavy or light chain constant domains. Human IgG heavy and light chain constant domain amino acid sequences are known in the art and are represented in table 1.

Table 1: sequences of human IgG heavy and light chain constant domains

Still further, a binding protein (e.g., an antibody) of the invention can be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of a binding protein of the invention to one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to make tetrameric scFv molecules (Kipriyanov et al, Human Antibodies and hybrids 6: 93-101, 1995), and the use of cysteine residues, a tag peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al, mol. Immunol. 31: 1047-1058, 1994). Antibody moieties such as Fab and F (ab') ₂ Fragments may be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies, respectively. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained as described herein using standard recombinant DNA techniques.

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigenic specificities. However, an isolated antibody that specifically binds to an A β (20-42) globulomer may have cross-reactivity with other antigens, such as an A β globulomer, e.g., an A β (12-42) globulomer or other forms of A β. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals and/or any other targeted a β forms.

As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include, for example, amino acid residues in the CDRs, and in particular CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described in section B below), antibodies isolated from recombinant, combinatorial human antibody libraries (Hoogenboom, TIB technology 15: 62-70, 1997; Azzazy and Highsmith, Clin. biochem. 35: 425. 445, 2002; Gavido J. V. and Larrick J.W. (2002) Biotechnologies 29: 128. 145; Hoogboom H. and Chames P. (2000) Immunology Today 21: 371;. 378) antibodies isolated from animals transgenic for human immunoglobulin genes (e.g., mice) (see, e.g., Taylor, L. D. et al (1992) Aclock. Res. 6220: 6223. 35: 92. Biotechnology A) and Op # 629 5: 5987; Biotechnology A5923: 35. 76; and Biotechnology A # 629.), or by any other method involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo.

The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

The term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species, but in which the sequences of one or more CDR regions of VH and/or VL are replaced with CDR sequences of another species, for example an antibody having murine CDRs (e.g., CDR 3) in which one or more murine variable heavy and light chain regions have been replaced with human variable heavy and light chain sequences.

The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, as recognized in the art, refer to a numbering system of amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al (1971) Ann. NY Acad, Sci. 190: 382. 391 and Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Department of Health and Human Services, NIH publication No. 91-3242). The hypervariable region is amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3 for the heavy chain variable region. The hypervariable region has amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3 for the light chain variable region.

As used herein, the terms "acceptor" and "acceptor antibody" refer to an antibody or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. In some embodiments, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more constant regions. In another embodiment, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more framework regions and one or more constant regions. In particular embodiments, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. According to this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present at one or more specific positions of the human antibody. The acceptor framework region and/or the one or more acceptor constant regions may, for example, be derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., an antibody well known in the art, an antibody in development, or a commercially available antibody).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, named CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "set of CDRs" refers to a set of three CDRs present in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs which can be referred to as Kabat. Chothia and colleagues (Chothia & Lesk, J. mol. biol. 196: 901. 917 (1987) and Chothia et al, Nature 342: 877. Cho. 883 (1989)) found that certain sub-parts within the Kabat CDRs adopt almost identical peptide backbone conformations despite having a large diversity at the amino acid sequence level, these sub-parts are designated L1, L2 and L3 or H1, H2 and H3, where "L" and "L" overlap with other regions of the Kabat "heavy chain", respectively designated as H. kappa. 139 and H. kappa. 139 (1995) MacCallum (J Mol Biol 262 (5): 732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nevertheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even the entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, with particular embodiments using Kabat or Chothia defined CDRs.

As used herein, the term "canonical" residue refers to a residue that defines a particular canonical CDR structure in a CDR or framework, as defined by Chothia et al (J. mol. biol. 196: 901-. According to Chothia et al, the key portions of the CDRs of many antibodies have nearly identical peptide backbone conformations, despite the great diversity at the amino acid sequence level. Each canonical structure defines a set of peptide backbone torsion angles, primarily for contiguous segments of amino acid residues that form loops.

As used herein, the terms "donor" and "donor antibody" refer to an antibody that provides one or more CDRs. In one embodiment, the donor antibody is an antibody from a different species than the antibody from which the framework regions are obtained or derived. In the context of humanized antibodies, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs.

As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequences can be determined by different systems, the meaning of the framework sequences is interpreted correspondingly differently. The six CDRs (CDR-L1, -L2 and-L3 for the light chain and CDR-H1, -H2 and-H3 for the heavy chain) also split the framework regions on the light and heavy chains into four subregions on each chain (FR 1, FR2, FR3 and FR 4), with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. Specific subregions are not designated FR1, FR2, FR3 or FR4, as mentioned by others, and the framework regions represent combined FR's within a single naturally occurring immunoglobulin variable region. As used herein, FR represents one of the four subregions, and FRs represents two or more of the four subregions that make up the framework regions.

Human heavy and light chain acceptor sequences are known in the art. In one embodiment of the invention, the human heavy and light chain acceptor sequences are selected from the sequences described in table 2 and table 3. In another embodiment, the human heavy and light chain acceptor sequences are selected from sequences at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences set forth in table 2 and table 3.

Table 2: heavy chain acceptor sequence

Table 3: light chain acceptor sequences

As used herein, the term "germline antibody gene" or "gene segment" refers to immunoglobulin sequences encoded by non-lymphoid cells that have not undergone a maturation process that results in genetic rearrangements and mutations for expression of a particular immunoglobulin (see, e.g., Shapiro et al, Crit. Rev. Immunol. 22 (3): 183-200 (2002); Marchalonis et al, Adv Exp Med biol. 484:13-30 (2001)). One of the advantages provided by the various embodiments of the present invention stems from the recognition that: germline antibody genes are more likely than mature antibody genes to preserve the basic amino acid sequence structure unique to an individual in a species and therefore less likely to be identified as being from a foreign source when used therapeutically in that species.

As used herein, the term "key" residues refers to certain residues within the variable region that have more of an effect on the binding specificity and/or affinity of an antibody, particularly a humanized antibody. Key residues include, but are not limited to, one or more of the following: residues proximal to the CDRs, potential glycosylation sites (which may be N or O-glycosylation sites), rare residues, residues capable of interacting with antigen, residues capable of interacting with CDRs, canonical residues, contact residues between the heavy chain variable region and the light chain variable region, residues within the Vernier zone, and residues in the region that overlaps between the Chothia definition of the variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest and comprises a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of CDRs refers to CDRs having an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a nonhuman antibody CDR. Humanized antibodies comprise substantially all of at least one, and typically 2, variable domains (Fab, Fab ', F (ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody comprises a light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In a specific embodiment, the humanized antibody comprises only humanized variable domains of a light chain and/or a heavy chain.

Humanized antibodies may be selected from any class of immunoglobulin including IgM, IgG, IgD, IgA, and IgE, and any isotype including, but not limited to, IgG1, IgG2, IgG3, and IgG 4. Humanized antibodies may include sequences from more than one species or isotype, and specific constant domains may be selected using techniques well known in the art to optimize desired effector function.

The framework and CDR regions of the humanized antibody need not correspond exactly to the parental sequences, e.g., donor antibody CDRs, or the consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDRs or framework residues at that position do not correspond to the donor antibody or the consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to a framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, germany 1987)). In the immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

As used herein, the "Vernier" region refers to a subset of framework residues that can modulate the CDR structure and fine tune the fit (fit) to the antigen, as described by Foote and Winter (1992, J. mol. biol.224:487-499, which is incorporated herein by reference). Vernier zone residues form the basis layer of CDRs and can influence the structure of CDRs and the affinity of antibodies.

As used herein, the term "antibody" also encompasses multivalent binding proteins. The term "multivalent binding protein" is used in this specification to indicate a binding protein comprising two or more antigen binding sites. Multivalent binding proteins are engineered to have three or more antigen binding sites and are not generally naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein capable of binding two or more related or unrelated targets. A Dual Variable Domain (DVD) binding protein as used herein is a binding protein that comprises two or more antigen binding sites and is a tetravalent or multivalent binding protein. Such DVDs can be monospecific, i.e., capable of binding one antigen, or multispecific, i.e., capable of binding two or more antigens. A DVD-binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as DVD-Ig. Each half of the DVD-Ig contains a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain, wherein each antigen binding site has a total of 6 CDRs associated with antigen binding. DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. patent application No. 11/507,050 and are incorporated herein by reference.

The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings (groupings) of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes are regions of an antigen that are bound by a binding protein, particularly an antibody. In certain embodiments, a binding protein or antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

The binding affinity of the antibodies of the invention can be assessed by using standardized in vitro immunoassays, such as ELISA, dot blot or BIAcore assays (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see subson, u., et al (1993) ann. biol. clin. 51: 19-26; j insson, U.S., et al (1991) Biotechniques 11: 620-; johnsson, B.et al (1995) J. mol. Recognit. 8: 125-131; and Johnsson, B., et al (1991) anal. biochem. 198: 268. sup. 277.

According to a particular embodiment, affinity as defined herein refers to the value obtained by performing a dot blot and evaluating it by densitometry. According to a particular embodiment of the invention, the determination of the binding affinity by dot blot comprises the following: a specific amount of antigen (e.g. a β (X-Y) globulomer, a β (X-Y) monomer or a β (X-Y) filament as defined above), or conveniently, e.g. in 20 mM NaH2PO4, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA to a suitable dilution of the antigen concentration of e.g. 100 pmol/μ l, 10 pmol/μ l, 1 pmol/μ l, 0.1 pmol/μ l and 0.01 pmol/μ l, is spotted on a cellulose membrane, followed by blocking the membrane with milk to prevent non-specific binding, and washing, followed by contact with the antibody of interest, followed by detection of the latter by means of an enzyme-conjugated secondary antibody and a colorimetric reaction; at defined antibody concentrations, the amount of antibody bound allows for an affinity assay. Thus, the relative affinities of 2 different antibodies to 1 target or 1 antibody to 2 different targets are defined herein as the relationship of the respective amounts of target-bound antibody observed with 2 antibody-target combinations under otherwise identical dot blot conditions. Unlike similar methods based on western blotting, the dot blot method will determine the affinity of an antibody for a given target in its native conformation; unlike the ELISA method, the dot blot method does not have differences in affinity between different targets and the matrix, allowing for more accurate comparisons between different targets.

As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, sweden and Piscataway, NJ). For further description, see subson, u., et al (1993) ann. biol. clin., 51: 19-26; nanosenson et al, (1991) BioTechniques, 11: 620-627; johnsson et al, (1995) j. mol. recognit, 8: 125-131; and Johnnson et al (1991) anal. biochem, 198: 268-277.

As known in the art, the term "k" as used herein _on "(likewise," Kon "," Kon "," K ") _on ") means the binding rate constant of a binding protein (e.g., an antibody) binding to an antigen to form a binding complex, e.g., an antibody/antigen complex. Will also be "k _on "referred to by the term" association rate constant "Or "ka," as used interchangeably herein. This value is indicative of the binding rate of a binding protein (e.g., an antibody) to its target antigen, or the rate of complex formation between a binding protein (e.g., an antibody) and an antigen, as represented by the following equation:

Antibody ("Ab") + antigen ("Ag") → Ab-Ag.

As known in the art, the term "k" as used herein _off "(likewise," Koff "," K _off ") means the dissociation rate constant or" dissociation rate constant "for dissociation of a binding protein (e.g., an antibody) from a binding complex (e.g., an antibody/antigen complex). This value indicates the off-rate of a binding protein (e.g., antibody) from its target antigen, or the off-rate of separation of the Ab-Ag complex over time into free antibody and antigen, which is expressed as the following equation:

Ab + Ag ← Ab-Ag。

the term "K" as used herein _D "(likewise," K) _d "or" KD ") means" equilibrium dissociation constant "and means at equilibrium in a titration measurement, or by plotting the dissociation rate constant (k) _off ) Divided by the binding rate constant (k) _on ) The obtained value. Using the binding Rate constant (k) _on ) Dissociation rate constant (k) _off ) And equilibrium dissociation constant (K) _D ) Indicating the binding affinity of the binding protein (e.g., antibody) for the antigen. Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine the sample at equilibrium in physiological buffer. Other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) may be used for the determination (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, KinExA @ (dynamic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho) may also be used for the determination.

The term "labeled binding protein" as used herein refers to a binding protein with incorporation of a label, which is a knotPreparation was made for the identification of the complex protein. Likewise, the term "labeled antibody" as used herein refers to an antibody having incorporated a label that provides for the identification of the antibody. In one aspect, the label is a detectable label, e.g., incorporating a radiolabeled amino acid or attaching a biotinylated moiety to the polypeptide, which biotinylated moiety can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g., ³ H、 ¹⁴ C、 ³⁵ S、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I、 ¹⁷⁷ Lu、 ¹⁶⁶ ho or ¹⁵³ Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); a chemiluminescent label, a biotinylation group, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for a second antibody, metal binding domain, epitope tag), and a magnetic reagent such as gadolinium chelate.

As used herein, the term "antibody" also encompasses antibody conjugates. The term "antibody conjugate" refers to a binding protein, e.g., an antibody, chemically linked to a second chemical moiety, e.g., a therapeutic agent.

The term "therapeutic agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from a biological material that is a "cognitive enhancer drug," which is a drug that improves impaired human brain cognitive ability (i.e., thinking, learning, and memory). Cognitive enhancing drugs act by altering the availability of neurochemicals (e.g., neurotransmitters, enzymes and hormones), improving oxygen supply, stimulating nerve growth or inhibiting nerve damage. Examples of cognition enhancing agents include compounds that increase the activity of acetylcholine such as, but not limited to, acetylcholine receptor agonists (e.g., nicotinic alpha-7 receptor agonists)An agent or allosteric modulator, an alpha 4 beta 2 nicotinic receptor agonist or allosteric modulator), an acetylcholinesterase inhibitor (e.g., donepezil, rivastigmine, and galantamine), a butyrylcholinesterase inhibitor, an N-methyl-D-aspartate (NMDA) receptor antagonist (e.g., memantine), an activity-dependent neuroprotective protein (ADNP) agonist, a serotonin 5-HT 1A receptor agonist (e.g., zalioden), a 5-HT ₄ Receptor agonists, 5-HT ₆ Receptor antagonists, serotonin 1A receptor antagonists, histamine H ₃ Receptor antagonists, calpain inhibitors, Vascular Endothelial Growth Factor (VEGF) proteins or agonists, trophic growth factors, anti-apoptotic compounds, AMPA-type glutamate receptor activators, L-type or N-type calcium channel blockers or modulators, potassium channel blockers, hypoxia-inducible factor (HIF) activators, HIF prolyl 4-hydroxylase inhibitors, anti-inflammatory agents, inhibitors of amyloid a β peptide or amyloid plaques, tau hyperphosphorylation inhibitors, phosphodiesterase 5 inhibitors (e.g., tadalafil, sildenafil), phosphodiesterase 4 inhibitors, monoamine oxidase inhibitors, or pharmaceutically acceptable salts thereof. Specific examples of such cognitive enhancing drugs include, but are not limited to, cholinesterase inhibitors such as donepezil (Aricept) ^® ) Rivastigmine (Exelon) ^® ) Galantamine (remininyl) ^® ) N-methyl-D-aspartate antagonists such as memantine (Namenda) ^® ）。

As used herein, the terms "crystal" and "crystallized" refer to a binding protein (e.g., an antibody or antigen-binding portion thereof) that is present in crystal form. A crystal is a form of a solid state of matter that is different from other forms such as an amorphous solid state or a liquid crystal state. Crystals are composed of regular, repetitive, three-dimensional arrangements of atoms, ions, molecules (e.g., proteins such as antibodies), or combinations of molecules (e.g., antigen/antibody complexes). These three-dimensional arrangements are arranged according to specific mathematical relationships well known in the art. The repeating basic unit or building block in a crystal is called an asymmetric unit. The repetition of asymmetric units in an arrangement that conforms to a given, well-defined crystallographic symmetry provides a "unit cell" of the crystal. The crystals are provided by the repetition of unit cells regularly translated in all 3 dimensions. See, Giege, R. and Ducruix, A. Barrett, crystalization of Nucleic Acids and Proteins, a Practical Approach, 2 nd edition, pages 201-16, Oxford University Press, New York, New York, (1999) ".

As used herein, the term "neutralization" refers to the neutralization of a biological activity of a β form that is targeted when a binding protein specifically binds to the a β form. For example, a neutralizing binding protein is a neutralizing antibody whose binding to the a β (20-42) amino acid region of the globulomer (and/or any other targeted a β form) results in inhibition of the biological activity of the globulomer. According to one aspect of the invention, the neutralizing binding protein binds to the a β (20-42) region of the globulomer (and/or any other targeted a β form) and reduces the biological activity of the targeted a β form by at least about 20%, 40%, 60%, 80%, 85% or more. Biological activity targeting the a β form inhibition of binding proteins by neutralization can be assessed by measuring one or more indicators well known in the art that target the biological activity of the a β form, e.g., interaction (e.g., binding) of the a β form with P/Q type voltage-gated presynaptic calcium channels, inhibition of P/Q type voltage-gated presynaptic calcium channel activity, Ca through P/Q type voltage-gated presynaptic calcium channels ⁺⁺ Flux, local (e.g. intracellular) Ca ⁺⁺ Concentration, synaptic activity.

The term "activity" includes activities such as binding specificity/affinity of a binding protein, particularly an antibody, to an antigen such as the a β (20-42) globulomer (and any other targeted a β forms); and/or neutralizing potency of an antibody, e.g., an antibody whose binding to a targeted a β form inhibits the biological activity of the targeted a β form. The biological activity targeting the a β form comprises interaction of the a β form with P/Q type voltage-gated presynaptic calcium channels, which results in inhibition of the calcium channel activity.

The invention also provides isolated nucleotide sequences encoding the binding proteins of the invention. The present invention provides those nucleotide sequences (or fragments thereof) having a sequence that comprises, corresponds to, is equivalent to, is hybridizable, or complementary to, at least about 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), or at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to these encoding nucleotide sequences. (all integers (and parts thereof) between and including 70% and 100% are considered within the scope of the invention in terms of percent identity). Such sequences may be derived from any source (e.g., isolated from a natural source, produced via a semi-synthetic route, or newly synthesized). In particular, such sequences may be isolated or derived from sources other than those described in the examples (e.g., bacteria, fungi, algae, mouse, or human).

For the purposes of the present invention, a "fragment" of a nucleotide sequence is defined as a contiguous sequence of approximately at least 6, e.g., at least about 8, at least about 10, or at least about 15 nucleotides corresponding to a region of the specified nucleotide sequence.

The term "identity" refers to the relatedness of two sequences on a nucleotide-by-nucleotide basis over a particular comparison window or segment. Identity is therefore defined as the degree of identity, correspondence or equivalence between the identical strands (sense or antisense) of two DNA segments (or two amino acid sequences). "percent sequence identity" is calculated by: the two optimally aligned sequences are compared over a particular region, the number of positions at which equivalent bases or amino acids appear in the two sequences is determined in order to obtain the number of matching positions, such number of positions is divided by the total number of positions in the segment to be compared, and the result is multiplied by 100. Optimal alignment of sequences can be performed by: smith & Waterman, appl. Math. 2: 482, 1981, Needleman & Wunsch, j. mol. biol. 48: 443, 1970, Pearson & Lipman, proc. natl. acad. Sci. (USA) 85: 2444, 1988, and Computer programs for implementing the algorithms concerned (e.g., Cluster Macaw Pileup (http:// cmgm. stanford. edu/biochem218/11multiple. pdf; Higgins et al, CABIOS. 5L151-153, 1989), FASTDB (Intelligentics), BLAST (National Center for biological Information; Altschul et al, Nucleic Acids Research 25: 3389-3402, 1997), PILEUP (Genetics Computer Group, Madison, Wis.), or GAP, BESTFIT, FASTA and TFASTA (Wisconsics Software Package Release 7.0, Genetics Computer, Wis.). (see U.S. patent No. 5,912,120).

For the purposes of the present invention, "complementarity" is defined as the degree of relatedness between two DNA segments. It is determined by measuring the ability of the sense strand of one DNA segment to hybridize to the antisense strand of another DNA segment to form a duplex under suitable conditions. "complement" is defined as a sequence that pairs with a given sequence based on canonical base pairing rules. For example, the sequence A-G-T in one nucleotide strand is "complementary" to T-C-A in the other strand. In the double helix, adenine is present in one strand and thymine is present in the other strand. Similarly, whenever guanine is found in one strand, cytosine is found in the other. The greater the association between the nucleotide sequences of two DNA segments, the greater the ability to form a hybrid duplex between the strands of the two DNA segments.

"similarity" between two amino acid sequences is defined as the presence of a series of identical and conserved amino acid residues in the two sequences. The higher the degree of similarity between two amino acid sequences, the higher the correspondence, identity or equivalence of the two sequences. ("identity between two amino acid sequences is defined as the presence of a series of exactly identical or unchanged amino acid residues in the two sequences)," complementarity "," identity "and" similarity "are well known to those of ordinary skill in the art.

"encoded by … …" refers to a nucleic acid sequence that encodes a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 amino acids, e.g., at least 8 amino acids or at least 15 amino acids, from the polypeptide encoded by the nucleic acid sequence.

The term "polynucleotide" as referred to herein means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides (deoxynucleotides), or a modified form of either type of nucleotide. The term includes DNA in both single and double stranded form, but preferably is double stranded DNA.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, is "isolated polynucleotide": does not bind to all or a portion of the polynucleotide to which the "isolated polynucleotide" is found bound in nature; operably linked to a polynucleotide to which it is not linked in nature; or not as part of a larger sequence in nature.

As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and, thus, are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are typically in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functions.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "operably linked" sequences include expression control sequences that are contiguous with the gene of interest, and expression control sequences that function in trans or remotely to control the gene of interest. As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

As defined herein, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using a variety of methods well known in the art. Transformation may rely on any known method for inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication as an autonomously replicating plasmid or as part of the host chromosome. They also include cells that transiently express the inserted DNA or RNA for a limited period of time.

As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which exogenous DNA has been introduced. It is understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one aspect, host cells include prokaryotic and eukaryotic cells selected from any kingdom of life. Eukaryotic cells include protists, fungi, plant and animal cells. In another aspect, host cells include, but are not limited to, the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK 293 and COS; insect cell line Sf 9; and the fungal cell Saccharomyces cerevisiae.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly done in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, for example, Sambrook et al Molecular Cloning, which is incorporated herein by reference for any purpose: a Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).

As known in the art and as used herein, a "transgenic organism" refers to an organism having cells comprising a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide that is not naturally expressed in the organism. A "transgene" is a DNA construct that is stably and operably integrated into the genome of a cell from which the transgenic organism develops, thereby directing expression of the encoded gene product in one or more cell types or tissues of the transgenic organism.

The terms "modulate" and "tune" are used interchangeably and, as used herein, refer to a change or alteration in the activity of a molecule of interest (e.g., a biological activity that targets the a β form). Tuning can be an increase or decrease in a certain activity or functional magnitude of the molecule of interest. Exemplary activities and functions of the molecules include, but are not limited to, binding characteristics, enzymatic activity, cellular receptor activation, and signal transduction.

Accordingly, as used herein, the term "modulator" is a compound that is capable of modifying or altering the activity or function of a molecule of interest (e.g., targeting a biological activity of the a β form). For example, a modulator may cause an increase or decrease in the magnitude of an activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor that reduces the magnitude of at least one activity or function of a molecule.

As used herein, the term "agonist" refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of an activity or function of the molecule as compared to the magnitude of the activity or function observed in the absence of the agonist.

As used herein, the term "antagonist" or "inhibitor" refers to a modulator that, when contacted with a molecule of interest, causes a decrease in a certain activity or functional magnitude of the molecule as compared to the activity or functional magnitude observed in the absence of the antagonist. Antagonists of particular interest include those that block or modulate the biological activity of the targeted a β form. Antagonists and inhibitors targeting the a β form may include, but are not limited to, the binding proteins of the present invention, which bind to a β (20-42) globulomers and any other targeted a β forms. Antagonists or inhibitors targeting the a β form may, for example, reduce the inhibitory effect of the a β form on P/Q type voltage-gated presynaptic calcium channel activity.

As used herein, the term "effective amount" refers to an amount of a therapy sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the progression of a disorder, cause regression of a disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or ameliorate the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent).

As used herein, the term "sample" is used in its broadest sense. As used herein, a "biological sample" includes, but is not limited to, any amount of material from an organism (living thing) or from a precursor. Such organisms include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.

I. Antibodies of the invention

A first particular aspect of the invention provides an antibody or antigen-binding portion thereof that binds to an a β (20-42) globulomer and/or any other CDR-grafted targeted form of a β. A second particular aspect of the invention provides a humanized antibody or antigen-binding portion thereof that binds to the A β (20-42) globulomer and/or any other targeted A β form. According to a particular aspect, the antibody or portion thereof is an isolated antibody. According to a further particular aspect, the antibodies of the invention neutralize the activity of a β (20-42) globulomers and/or any other targeted a β forms.

A. Method for preparing anti-Abeta (20-42) globulomer antibody

Antibodies of the invention can be prepared by any of a number of techniques known in the art.

1. anti-Abeta (20-42) globulomer monoclonal antibodies using hybridoma technology

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal Antibodies can be produced using hybridoma technology, including those known in the art and exemplified by Harlow et al, Antibodies: a Laboratory Manual (Cold Spring Harbor Laboratory Press, 2 nd edition, 1988); hammerling, et al: monoconal Antibodies and T-Cell hybrids 563-681 (Elsevier, N.Y., 1981), which is incorporated by reference in its entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, rather than the method by which it was produced.

Methods for generating and screening specific antibodies using hybridoma technology are routine and well known in the art. In one embodiment, the invention provides a method of producing monoclonal antibodies and antibodies produced by the method, comprising culturing hybridoma cells that secrete an antibody of the invention, wherein, for example, hybridomas are produced by fusing spleen cells isolated from mice immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete antibodies capable of binding a polypeptide of the invention. Briefly, mice can be immunized with A β (20-42) globulomer antigen. In particular embodiments, the antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a localized deposit, or they may comprise substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is to be administered, the immunization schedule will involve 2 or more administrations of the polypeptide spread out over several weeks.

After immunization of an animal with the A β (20-42) globulomer antigen, antibodies and/or antibody producing cells can be obtained from the animal. Sera containing anti-A β (20-42) globulomer antibodies were obtained from animals by exsanguinating or sacrificing the animals. The serum may be used as it is obtained from an animal, immunoglobulin fractions may be obtained from the serum, or anti-a β (20-42) globulomer antibodies may be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal and thus has the characteristics of a heterogeneous array.

Once an immune response is detected, e.g., antibodies specific for the antigen A β (20-42) globulomer are detected in mouse serum, mouse spleens are harvested and splenocytes isolated. The splenocytes are then fused by well known techniques with any suitable myeloma cells, such as cells from cell line SP20 available from ATCC. Hybridomas were selected and cloned by limiting dilution. Hybridoma clones were then assayed by methods known in the art for cells that secrete antibodies capable of binding to the A β (20-42) globulomer. Ascites fluid, which typically contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas can be produced from immunized animals. Following immunization, the animals are sacrificed and splenic B cells are fused with immortal myeloma cells, as is well known in the art. (see, e.g., Harlow and Lane, supra). In a particular embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, hybridomas are screened using A β (20-42) globulomers or portions thereof or cells expressing A β (20-42) globulomers. In particular embodiments, the initial screening is performed using enzyme-linked immunoassays (ELISA) or Radioimmunoassays (RIA). Examples of ELISA screens are provided in WO 00/37504, which is incorporated herein by reference.

As discussed further below, anti-a β (20-42) globulomer antibody-producing hybridomas are selected, cloned, and further screened for desired characteristics, including strong hybridoma growth, high antibody production, and desired antibody characteristics. Hybridomas can be cultured and expanded in syngeneic animals, in animals lacking the immune system, such as nude mice, or in cell culture in vitro. Methods for selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

In a particular embodiment, as described above, the hybridoma is a mouse hybridoma. In another specific embodiment, the hybridoma is produced in a non-human, non-mouse species, such as rat, sheep, pig, goat, cow, or horse. In another embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused to a human cell expressing an anti-A β (20-42) globulomer antibody.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F (ab ') 2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 fragments). The F (ab') 2 fragment comprises the variable region, the light chain constant region, and the CH1 domain of the heavy chain.

2. anti-Abeta (20-42) globulomer monoclonal antibodies using SLAM

In another aspect of the invention, recombinant antibodies are generated from individual, isolated lymphocytes using a procedure known in the art as the Selective Lymphocyte Antibody Method (SLAM) as described in U.S. Pat. No. 5,627,052, PCT publication WO92/02551 and Babcock, J.S. et al (1996) Proc. Natl. Acad. Sci. USA 93: 7843-. In this method, antigen-specific hemolytic plaque assays are used to screen individual cells secreting an antibody of interest, such as lymphocytes derived from any of the immunized animals described in section 1, where the antigen a β (20-42) globulomer or subunit thereof is coupled to sheep red blood cells using a linker such as biotin, and used to identify individual cells secreting an antibody specific for a β (20-42) globulomer. After the antibody-secreting cells of interest are identified, the heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase PCR, and these variable regions can then be expressed in a mammalian host cell, such as COS or CHO cells, in a suitable background of immunoglobulin constant regions (e.g., human constant regions). Host cells transfected with amplified immunoglobulin sequences derived from lymphocytes selected in vivo may then be subjected to further in vitro analysis and selection, for example by panning the transfected cells to isolate cells expressing antibodies against a β (20-42) globulomers. The amplified immunoglobulin sequences may further be subjected to in vitro processing, for example by in vitro affinity maturation methods, such as those described in PCT publication WO 97/29131 and PCT publication WO 00/56772.

3. anti-Abeta (20-42) globulomer monoclonal antibodies using transgenic animals

In another embodiment of the invention, antibodies are produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with an A β (20-42) antigen. In particular embodiments, the non-human animal is a XENOMOUSE transgenic mouse, an engineered mouse strain comprising a large fragment of a human immunoglobulin locus and defective in mouse antibody production. See, e.g., Green et al, Nature Genetics 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741 published on

month

7 and 25 of 1991, WO 94/02602 published on

month

2 and 3 of 1994, WO 96/34096 and WO 96/33735 both published on month 10 and 31 of 1996, WO 98/16654 published on

month

4 and 23 of 1998, WO 98/24893 published on month 11 of 1998, WO 98/50433 published on

month

11 and 12 of 1998, WO 99/45031 published on

month

9 and 10 of 1999, WO 99/53049 published on

month

10 and 21 of 1999, WO 00/09560 published on

month

2 and 24 of 2000, and WO 00/037504 published on

month

6 and 29 of 2000. The XENOMOUSE transgenic mice produce a whole human antibody adult-like repertoire and produce antigen-specific human monoclonal antibodies. XENOMOUSE transgenic mice contain an approximately 80% human antibody profile by introducing megabase-sized, germline-configured YAC fragments of the human heavy chain locus and the x light chain locus. See Mendez et al, Nature Genetics 15: 146-.

4. anti-A beta (20-42) globulomer monoclonal antibodies using recombinant antibody libraries

In vitro methods may also be used to prepare antibodies of the invention, wherein a library of antibodies is screened to identify antibodies with the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include those described in the following references: for example, U.S. Pat. Nos. 5,223,409 to Ladner et al; kang et al PCT publication No. WO 92/18619; dower et al PCT publication Nos. WO 91/17271; winter et al PCT publication No. WO 92/20791; markland et al PCT publication No. WO 92/15679; breitling et al PCT publication No. WO 93/01288; PCT publication No. WO92/01047 to McCafferty et al; garrrard et al PCT publication No. WO 92/09690; fuchs et al (1991) Bio/Technology 9: 1370-; hay et al (1992) Hum antibody hybrids 3: 81-85; huse et al (1989) Science 246: 1275-1281; McCafferty et al, Nature (1990) 348: 552-554; griffiths et al (1993) EMBO J12: 725-doped 734; hawkins et al (1992) J Mol Biol 226: 889-896; clackson et al (1991) Nature 352: 624-; gram et al (1992) PNAS 89: 3576-3580; garrad et al (1991) Bio/Technology 9: 1373-1377; hoogenboom et al (1991) Nuc Acid Res 19:4133 and 4137; and Barbas et al (1991) PNAS 88: 7978-.

The library of recombinant antibodies may be from a subject immunized with an A β (20-42) globulomer or a portion of an A β (20-42) globulomer. Alternatively, the library of recombinant antibodies may be from a subject used for the first time in an experiment, i.e. a subject that has not been immunized with an a β (20-42) globulomer, for example a library of human antibodies from a human subject that has not been immunized with an a β (20-42) globulomer. Antibodies of the invention are selected by screening a recombinant antibody library with peptides comprising human A β (20-42) globulomers to thereby select those antibodies that recognize A β (20-42) globulomers and distinguish A β (1-42) globulomers, A β (1-40) and A β (1-42) monomers, A β fibrils, and sAPP α. Methods for performing such screening and selection are well known in the art, for example as described in the references in the previous paragraphs. To select antibodies of the invention that have a particular binding affinity for the A β (20-42) globulomer and distinguish A β (1-42) globulomer, A β (1-40) and A β (1-42) monomers, A β fibrils, and sAPP α, such as those that dissociate from human A β (20-42) globulomer with a particular koff rate constant, dot blot methods known in the art may be used to select antibodies with the desired koff rate constant. To select antibodies of the invention, such as those with a specific IC50, that have a specific neutralizing activity for the a β (20-42) globulomer and distinguish between the a β (1-42) globulomer, the a β (1-40) and a β (1-42) monomers, the a β fibril, and the sAPP α, standard methods known in the art for assessing inhibition of a β (20-42) globulomer activity can be used.

In one aspect, the invention relates to an isolated antibody or antigen-binding portion thereof that binds to a human A β (20-42) globulomer and distinguishes A β (1-42) globulomer, A β (1-40) and A β (1-42) monomers, A β fibrils, and sAPP α. According to one aspect, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the invention can also be produced using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, such phage can be used to display antigen binding domains expressed by a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds an antigen of interest can be selected or identified with the antigen, for example using a labeled antigen or an antigen bound or captured by a solid surface or bead. The phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed by phage having Fab, Fv, or dsFv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies of the invention include those disclosed in the following references: brinkman et al, J. Immunol. Methods 182:41-50 (1995); ames et al, J. Immunol. Methods 184:177-186 (1995); kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); persic et al, Gene 1879-18 (1997); burton et al, Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB 91/01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753, respectively; 5,821,047, respectively; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727, respectively; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the references above, following phage selection, the antibody coding regions from the phage can be isolated and used to produce whole antibodies, including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, techniques for the recombinant production of Fab, Fab 'and F (ab') 2 fragments may also be employed, using methods known in the art, such as those disclosed in the following references: PCT publication WO 92/22324; mullinax et al, BioTechniques 12 (6): 864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240: 1041-. Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in the following references: U.S. Pat. nos. 4,946,778 and 5,258,498; huston et al, Methods in Enzymology 203:46-88 (1991); shu et al, PNAS 90:7995-7999 (1993); and Skerra et al, Science 240: 1038-.

As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can be applied to the identification of dual specific antibodies of the invention. Such as PCT publication No. WO 98/31700 to Szostak and Roberts, r.w. and Szostak, J.W, (1997) proc. natl. acad. sci. USA, 94: an alternative expression system is described in 12297-12302, where a library of recombinant antibodies is expressed as RNA-protein fusions. In this system, a covalent fusion is created between the mRNA and its encoded peptide or protein by in vitro translation of synthetic mRNAs carrying a puromycin-peptidyl acceptor antibiotic at their 3' end. Thus, based on the nature of the encoded peptide or protein, e.g., antibody or portion thereof, e.g., binding of the antibody or portion thereof to a dual specific antigen, a particular mRNA can be enriched from a complex mixture (e.g., combinatorial library) of mRNAs. The nucleic acid sequences encoding the antibodies or portions thereof recovered from such library screening may be expressed by recombinant methods as described above (e.g., in mammalian host cells), and further affinity maturation may be carried out by additional screening cycles of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence or sequences, or by other methods described above for in vitro affinity maturation of recombinant antibodies.

In another approach, the antibodies of the invention can also be produced using yeast display methods known in the art. In the yeast display method, using genetic methods to make antibody domains bound to the yeast cell wall, and they are displayed on the yeast surface. In particular, such yeast can be used to display by the spectrum or combinatorial antibody library (e.g., human or murine) expression of antigen binding domains. Examples of yeast display methods that may be used to prepare the antibodies of the invention include those disclosed in Wittrup et al, U.S. patent No. 6,699,658, which is incorporated herein by reference.

B. Generation of recombinant Abeta (20-42) globulomer antibodies

The antibodies of the invention can be produced by any of a number of techniques known in the art. For example, expression from a host cell, wherein one or more expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate precipitation, DEAE dextran transfection, and the like. The antibodies of the invention may be expressed in prokaryotic or eukaryotic host cells. According to a particular aspect of the invention, expression of the antibody is performed using eukaryotic cells, such as mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies.

According to one aspect, mammalian host cells for expression of the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (including those described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA, 77: 4216-. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient period of time to allow expression of the antibody in the host cell, or secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be appreciated that variations on the above procedures are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light and/or heavy chain of an antibody of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed by such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies can be produced by cross-linking an antibody of the invention with a second antibody via standard chemical cross-linking methods, wherein one heavy chain and one light chain is an antibody of the invention, and the other heavy and light chains are specific for an antigen other than the antigen of interest.

In a particular system for recombinant expression of the antibodies or antigen-binding portions thereof of the invention, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene that allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow expression of antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the antibodies from the culture medium. Still further, the present invention provides a method of synthesizing the recombinant antibody of the present invention by culturing the host cell of the present invention in a suitable medium until the recombinant antibody of the present invention is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

1. anti-Abeta (20-42) globulomer murine antibodies

Table 4 is a listing of the amino acid sequences of the VH and VL regions of murine 4D 10.

Table 4: list of amino acid sequences of VH and VL regions

CDRs are underlined in murine light and heavy chains.

2. anti-A beta (20-42) globulomer chimeric antibodies

Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, for example, antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art and discussed in detail herein. See, e.g., Morrison, Science 229:1202 (1985); oi et al, BioTechniques 4:214 (1986); gillies et al, (1989) J. Immunol. Methods 125: 191-202; U.S. patent nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Furthermore, techniques developed for the production of "chimeric antibodies" (Morrison et al, 1984, Proc. Natl. Acad. Sci. 81: 851-855; Neuberger et al, 1984, Nature 312: 604-608; Takeda et al, 1985, Nature 314:452-454, which is incorporated herein by reference in its entirety) can be used, which are achieved by splicing genes from mouse antibody molecules with the appropriate antigen specificity together with genes from human antibody molecules with the appropriate biological activity.

In one embodiment, the chimeric antibody of the invention is produced by replacing the heavy chain constant region of the murine monoclonal anti-a β (20-42) globulomer antibody 4D10 described in WO2007/062852 with a human IgG1 constant region.

3. anti-A beta (20-42) globulomer CDR grafted antibodies

The CDR-grafted antibodies of the invention comprise heavy and light chain variable region sequences from human antibodies, wherein one or more CDR regions of VH and/or VL are replaced with CDR sequences of the murine antibodies of the invention. The framework sequences from any human antibody can serve as a template for CDR grafting. However, direct chain replacement on such frameworks often results in some loss of binding affinity to the antigen. The more homologous a human antibody is to the original murine antibody, the less likely it is that combining murine CDRs with a human framework will introduce a distortion in the CDRs that can reduce affinity. Thus, the human variable framework selected to replace the murine variable framework except for the CDRs has, for example, at least 65% sequence identity with the murine antibody variable region framework. The human and murine variable regions, except for the CDRs, have, for example, at least 70%, at least 75%, or at least 80% sequence identity. Methods for producing chimeric antibodies are known in the art and are discussed in detail herein. (see also EP 239,400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539; 5,530,101; and 5,585,089), veneering (tunnelling) or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al, Protein Engineering 7 (6): 805-814 (1994); Roguska et al, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).

Table 5 below illustrates the sequences of the CDR-grafted antibody (4D 10hum antibody) of the present invention and the CDRs contained therein.

Table 5: amino acid sequence listing of VH and VL regions of CDR-grafted antibodies

CDRs are underlined in the humanized light and heavy chains.

4. anti-A beta (20-42) globulomer humanized antibodies

Humanized antibodies are antibody molecules derived from antibodies of a non-human species that bind the desired antigen, having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from human immunoglobulin molecules.

Known human Ig sequences are disclosed, for example,

each incorporated herein by reference in its entirety. Such imported sequences may be used to reduce immunogenicity, or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic, as is known in the art.

Framework residues in the human framework regions can be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example by modeling the interaction of the CDRs and framework residues to identify framework residues important for antigen binding, and sequence comparisons to identify rare framework residues at specific positions. See, e.g., Queen et al, U.S. patent nos. 5,585,089; riechmann et al, Nature, 332: 323-327 (1988), which is incorporated herein by reference in its entirety. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that exemplify and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These displayed examinations allow the analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from consensus and import sequences such that a desired antibody characteristic, such as increased affinity for one or more target antigens, is achieved. In general, CDR residues are directly and most importantly involved in affecting antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, those described in the following references: jones et al, Nature 321:522 (1986); verhoeyen et al, Science 239:1534 (1988), Sims et al, J. Immunol. 151: 2296 (1993); chothia and Lesk, J. mol. biol. 196:901 (1987), Carter et al, Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); presta et al, J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28 (4/5): 489-; studnicka et al, Protein Engineering 7 (6): 805-814 (1994); roguska et al, PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB 92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5814476, 5763192, 5723323, 5,766886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, each of which is incorporated herein by reference in its entirety, including the references cited therein.

Table 6 below illustrates the sequences of the humanized antibody (4D 10hum antibody) of the present invention and CDRs contained therein.

Table 6: amino acid sequence listing of VH and VL regions of humanized antibodies

CDRs are underlined in the humanized light and heavy chains.

C. Antibodies and antibody-producing cell lines

According to one aspect, the anti-a β (20-42) globulomer antibodies of the invention, or antibodies directed against any other targeted a β form, exhibit a high ability to reduce or neutralize the activity of a β (20-42) globulomer (and/or any other targeted a β form).

In particular embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. According to one aspect, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. According to a further aspect, the antibody comprises a light chain constant region, a kappa light chain constant region, or a lambda light chain constant region. According to one aspect, the antibody comprises a kappa light chain constant region. The antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.

Substitutions of amino acid residues in the Fc portion that alter antibody effector functions are known in the art (Winter et al, U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, Complement Dependent Cytotoxicity (CDC) and half-life/clearance of the antibody and antigen-antibody complex. Depending on the therapeutic purpose, in some cases these effector functions are desirable for therapeutic antibodies, but in other cases may be unnecessary or even detrimental. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to Fc γ Rs and complement C1q, respectively. Neonatal Fc receptor (FcRn) is a key component in determining the circulating half-life of an antibody. In another embodiment, at least one amino acid residue is substituted in an antibody constant region, such as the Fc region of an antibody, such that the effector function of the antibody is altered.

One embodiment provides a labeled antibody, wherein the antibody of the invention is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled antibody of the invention may be derivatized by functionally linking (by chemical coupling, genetic fusion, non-covalent binding, or otherwise) the antibody of the invention to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a pharmaceutical agent, and/or a protein or peptide that can mediate the binding of the antibody to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

Useful detectable reagents from which the antibodies of the invention can be derivatized include fluorescent compounds. Exemplary fluorescently detectable reagents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. The antibody may also be derivatized with a detectable enzyme, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, and the like. When an antibody is derivatized with a detectable enzyme, it is detected by the addition of an additional reagent for the production of a detectable reaction product by the enzyme. For example, when the detectable reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a detectable colored reaction product. Antibodies can also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized antibody. According to one aspect, the invention relates to crystals of intact anti-a β (20-42) globulomer antibodies and fragments thereof as disclosed herein, as well as formulations and compositions comprising such crystals. According to a further aspect, the crystallized antibody has a longer half-life in vivo than the soluble counterpart of the antibody. According to a further aspect, the antibody retains biological activity after crystallization.

The crystallized antibodies of the present invention may be produced according to methods known in the art and as disclosed in WO02/072636, which is incorporated herein by reference.

Another embodiment of the invention provides a glycosylated antibody, wherein the antibody comprises one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues can be added enzymatically, a process known as glycosylation. The resulting protein with covalently attached oligosaccharide side chains is called a glycosylated protein or glycoprotein.

Antibodies are glycoproteins having one or more carbohydrate residues in the Fc domain as well as in the variable domain. Carbohydrate residues in the Fc domain play an important role in the effector function of the Fc domain, with minimal effect on the antigen binding or half-life of the antibody (r. Jefferis, biotechnol. prog. 21 (2005), pages 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may have a negative effect on antibody binding affinity, possibly due to steric hindrance (Co, M.S., et al, mol. Immunol. (1993) 30: 1361. sup. 1367), or lead to an increased affinity for antigen (Wallick, S.C., et al, exp. Med. (1988) 168: 1099. sup. 1109; Wright, A., et al, EMBO J. (1991) 10: 27172723).

One aspect of the invention relates to the generation of glycosylation site mutants, wherein the O or N-linked glycosylation site of an antibody has been mutated. Such mutants can be generated by one skilled in the art using standard well-known techniques. The generation of glycosylation site mutants that retain biological activity but have increased or decreased binding activity is another object of the invention.

In another embodiment, the glycosylation of the antibody of the invention is modified. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) may be prepared. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. Such methods are described in further detail in international application publication No. WO03/016466a2 and U.S. patent nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

Additionally or alternatively, modified antibodies of the invention with altered glycosylation patterns can be prepared, such as hypofucosylated (hypofucosylated) antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNAc structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce antibodies with altered glycosylation. See, e.g., Shields, R.L. et al (2002) J. biol. chem. 277: 26733-26740; umana et al (1999) nat. Biotech.17: 176-1, and European patent Nos: EP1,176,195; international application publication Nos. WO03/035835 and WO 99/5434280, each of which is incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms can produce different glycosylases (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to such factors, protein glycosylation patterns and glycosyl residue compositions can vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful in the present invention can include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine, and sialic acid. According to one aspect, the glycosylated antibody comprises glycosyl residues such that the glycosylation pattern is human.

Different protein glycosylation can result in different protein characteristics, as is known to those skilled in the art. For example, the efficacy of a therapeutic protein produced in a microbial host, e.g., yeast, and glycosylated using the yeast endogenous pathway may be reduced as compared to that of the same protein expressed in a mammalian cell, e.g., a CHO cell line. Such glycoproteins may also be immunogenic in humans and exhibit a reduced in vivo half-life following administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and promote rapid clearance of proteins from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Thus, a practitioner may prefer a therapeutic protein having a particular glycosylation composition and pattern, e.g., a glycosylation composition and pattern that is identical or at least similar to that produced in human cells or species-specific cells of the intended subject animal.

Expression of a glycosylated protein different from that of the host cell may be accomplished by genetically modifying the host cell to express a heterologous glycosylase. Using techniques known in the art, practitioners can generate antibodies that exhibit glycosylation of human proteins. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylases such that the glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation equivalent to that of animal cells, particularly human cells (U.S. patent application publication Nos. 20040018590 and 20020137134; and WO 05/100584).

Another embodiment relates to anti-idiotype (anti-Id) antibodies specific for such antibodies of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that is generally associated with an antigen binding region of another antibody. anti-Id can be prepared by immunizing an animal with an antibody or CDR-containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. The anti-Id antibody may also be used as an "immunogen" to induce an immune response in another animal, thereby generating what is known as an anti-Id antibody.

Furthermore, one skilled in the art will recognize that a protein of interest may be expressed using a library of host cells that are genetically engineered to express various glycosylation enzymes such that the member host cells of the library produce the protein of interest with variant glycosylation patterns. The practitioner can then select and isolate a protein of interest with a particular novel glycosylation pattern. According to a further aspect, proteins with a particular selection of novel glycosylation patterns exhibit improved or altered biological properties.

D. Use of anti-A beta (20-42) globulomer antibodies

Knowing its ability to bind to the A β (20-42) globulomer, the anti-A β (20-42) globulomer antibodies of the invention or antibodies directed against any other targeted A β form may be used to detect A β (20-42) globulomer and/or any other targeted A β form (e.g., in a biological sample such as serum, CSF, brain tissue or plasma) using conventional methodsA standard immunoassay, such as enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting a β (20-42) globulomer and/or any other targeted a β form in a biological sample, comprising contacting the biological sample with an antibody of the invention and detecting the antibody bound or unbound to the a β (20-42) globulomer (and/or any other targeted a β form) to thereby detect the a β (20-42) globulomer and/or any other targeted a β form in the biological sample. The antibody is labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine (dichlorotriazinylamine) fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; and examples of suitable radioactive materials include ³ H、 ¹⁴ C、 ³⁵ S、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I、 ¹⁷⁷ Lu、 ¹⁶⁶ Ho or ¹⁵³ Sm。

As an alternative to labeled antibodies, A β (20-42) globulomer and/or any other targeted A β form may be assayed in biological fluids by competitive immunoassays using A β (20-42) globulomer standards labeled with a detectable substance and unlabeled anti-A β (20-42) globulomer antibodies. In this assay, a biological sample, a labeled a β (20-42) globulomer standard and an anti a β (20-42) globulomer antibody binding protein are combined, and the amount of labeled a β (20-42) globulomer standard bound to unlabeled antibody is determined. The amount of A β (20-42) globulomer and/or any other targeted A β form in the biological sample is inversely proportional to the amount of labeled A β (20-42) globulomer standard bound to the anti-A β (20-42) globulomer antibody.

According to one aspect of the invention, the antibodies of the invention are capable of neutralizing a β (20-42) globulomer activity and/or any other activity targeting a β form in vitro and in vivo. Thus, such antibodies of the invention may be used to inhibit (i.e., reduce) the activity of the a β (20-42) globulomer and/or the activity of any other targeted a β form, for example, in cell cultures comprising the a β (20-42) globulomer and/or any other targeted a β form, in human subjects, or in other mammalian subjects having a β (20-42) globulomer and/or any other targeted a β form to which the antibody of the invention is cross-reactive. In one embodiment, the invention provides a method for inhibiting (i.e., reducing) the activity of an a β (20-42) globulomer and/or the activity of any other targeted a β form, comprising contacting an a β (20-42) globulomer and/or any other targeted a β form with an antibody of the invention such that the activity of the a β (20-42) globulomer and/or the activity of any other targeted a β form is inhibited (i.e., reduced). For example, in a cell culture comprising or suspected of comprising a β (20-42) globulomer and/or any other form of targeted a β, the antibody of the invention may be added to the culture medium to inhibit (i.e., reduce) a β (20-42) globulomer activity and/or the activity of any other form of targeted a β in the culture.

In another embodiment, the present invention provides a method for inhibiting (i.e., reducing) targeted Α β form activity in a subject, advantageously a subject suffering from a disease or disorder in which said Α β form activity is detrimental or a disease or disorder or condition selected from: alpha 1-antitrypsin deficiency, C1-inhibitor deficiency angioedema, antithrombin deficiency thromboembolic disease, kuru, Creutzfeldt-Jakob disease/scrapie, bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker disease, familial fatal insomnia, Huntington's disease, spinocerebellar ataxia, Machado-Joseph atrophy, dentatorubral pallidoluysian, frontotemporal dementia, sickle cell anemia, labile hemoglobin inclusion body hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic AL amyloidosis, nodular AL amyloidosis, systemic AA amyloidosis, prostate amyloidosis, hemodialysis amyloidosis, hereditary (iceland) cerebrovascular disease, Huntington's disease, familial visceral amyloidosis, familial visceral polyneuropathy, Alzheimer's disease, Parkinson's, Familial visceral amyloidosis, senile systemic amyloidosis, familial amyloid neuropathy, familial cardiac amyloidosis, Alzheimer's disease, Down's syndrome, medullary thyroid carcinoma, and type 2 diabetes (T2 DM).

The invention provides methods for inhibiting (i.e., reducing) the activity of a targeted form of a β in a subject having such a disease or disorder, comprising administering to the subject an antibody of the invention, such that the activity of the form of a β in the subject is inhibited (i.e., reduced). In one aspect of the invention, the targeted a β form is a human a β form and the subject is a human subject. Alternatively, the subject may be a non-human mammal expressing APP or any form of a β, resulting in the production of a targeted form of a β to which the antibodies of the invention are capable of binding. Further, the subject can be a non-human mammal into which a targeted A β form has been introduced (e.g., by administering the targeted A β form or by expressing APP or any other A β form that results in the production of the targeted A β form.

Another embodiment is a method for inhibiting (i.e., reducing) the activity of a targeted a β form in a subject having an amyloidosis, such as alzheimer's disease or down syndrome.

Conditions in which the activity of the targeted a β form is detrimental include diseases and other conditions in which the presence of the targeted a β form in a subject suffering from the condition has been shown or suspected to be responsible for the pathophysiology of the condition or is suspected to be a factor contributing to the worsening of the condition. Thus, a disorder in which targeting the activity of the a β form is detrimental is one in which inhibition (i.e., reduction) of the activity of the a β form is expected to alleviate some or all of the symptoms and/or progression of the disorder. Such a disorder may be evidenced, for example, by an increase in the concentration of a targeted a β form in a biological fluid of a subject having the disorder (e.g., an increase in the concentration of a targeted a β form in the subject's serum, brain tissue, plasma, cerebrospinal fluid, etc.), which may be detected, for example, using an anti-a β (20-42) globulomer antibody as described above and/or an antibody directed against any other targeted a β form, or against any antibody of any a β form comprising a globulomer epitope with which an antibody of the invention is reactive. Non-limiting examples of conditions that can be treated with the antibodies of the invention include those conditions disclosed herein and those discussed below in the section on pharmaceutical compositions of the antibodies of the invention.

In another embodiment, the invention relates to a method for preventing the progression (e.g., worsening) of a disease condition disclosed herein. The method comprises administering to a subject (e.g., a mammal, e.g., a human) in need of such treatment a therapeutically effective amount of any of the binding proteins or antibodies as described herein. Alternatively, the method comprises administering to the subject a therapeutically effective amount of any of the proteins as described herein in combination with a therapeutically effective amount of at least one therapeutic agent.

In the methods described above for preventing the development or progression of a disorder described herein, one or more biomarkers, diagnostic tests, or a combination of biomarkers and diagnostic tests known to those of skill in the art can be used to determine (1) whether a subject is at risk of developing one or more of the disorders described herein; or (2) whether a disorder described herein has progressed (e.g., worsened) in a subject previously diagnosed with one or more of the above-mentioned disorders.

One or more biomarkers, diagnostic tests, or a combination of biomarkers and diagnostic tests known in the art can be used to identify a subject at risk of developing a condition described herein. Likewise, known in the art One or more biomarkers, diagnostic tests, or a combination of biomarkers and diagnostic tests can be used to determine the progression of a disease or condition in a subject who has been identified as having a disorder described herein. For example, one or more biomarkers, neuroimaging markers, or a combination of biological or neuroimaging markers (e.g., MRI, etc.) can be used to identify subjects at risk of developing alzheimer's disease, or for those subjects identified as having alzheimer's disease, progression of the disease. Biomarkers that can be examined include, but are not limited to, beta-amyloid _1-42 Tau, phosphorylated tau (p tau), plasma Abeta antibody, alpha-antichymotrypsin, amyloid precursor protein, APP isoform ratio in platelets, beta-secretase (also known as BACE), CD59, 8-hydroxy-deoxyguanine, glutamine synthetase, Glial Fibrillary Acidic Protein (GFAP), antibodies against GFAP, interleukin-6-receptor complex, vasopressin, melanotransferrin, neuromicrofilamentin, nitrotyrosine, hydroxysteroids, sulfatides, synaptic markers, S100 beta, NPS, plasma signaling proteins, and the like, or any combination thereof (see Shaw, L., et al, Nature Reviews2007, 6, 295-,Current Med. Chem. 2007，14，1171-1178.phillips, k., et al,Nature Reviews2006, 5463-,Neurology2007, 69, 1006-1011; ray, s, et al,Nature Medicine2007, 13 (11), 1359-, Neurology 2007，69，1622-1634.）。

E. pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the antibodies of the invention are useful for, but not limited to, diagnosing, detecting or monitoring a disorder, preventing, treating, managing or ameliorating and/or studying a disorder or one or more symptoms thereof. In particular embodiments, the compositions comprise one or more antibodies of the invention. In another embodiment, a pharmaceutical composition comprises one or more antibodies of the invention and, in addition to the antibodies of the invention, one or more prophylactic or therapeutic agents for the treatment of a disorder in which activity of a targeted a β form is detrimental. In a further embodiment, the prophylactic or therapeutic agent is known to be useful in the prevention, treatment, management or amelioration of the disorder or one or more symptoms thereof, or has been used therein or is currently being used therein. In accordance with these embodiments, the composition may further comprise a carrier, diluent or excipient.

The antibodies of the invention may be incorporated into pharmaceutical compositions suitable for administration to a subject. Generally, a pharmaceutical composition comprises an antibody of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.

In a further embodiment, the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder as described herein.

Various delivery systems are known and can be used to administer one or more antibodies of the invention or a combination of one or more antibodies of the invention and a prophylactic or therapeutic agent for the prevention, management, treatment or amelioration of a disorder or one or more symptoms thereof, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, j. biol. chem. 262:4429-4432 (1987)), nucleic acid constructs as part of a retrovirus or other vector, and the like. Methods of administering the prophylactic or therapeutic agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural (epidurala) administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration may be used, for example, using an inhaler or nebulizer, and formulations containing an aerosolizing agent (aerosol agent). See, e.g., U.S. patent nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT publication Nos. WO 92/19244, WO97/32572, WO97/44013, WO98/31346, and WO99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, the antibody of the invention, the combination therapy, or the composition of the invention is administered using Alkermes AIR lung drug delivery technology (Alkermes, inc., Cambridge, Mass). In specific embodiments, the prophylactic or therapeutic agent of the invention is administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonarily, or subcutaneously. The prophylactic or therapeutic agent may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered in conjunction with other biologically active agents. Administration may be systemic or local.

In particular embodiments, it may be desirable to have the antibodies of the invention administered locally to the area in need of treatment; this may be accomplished, for example, but not limited to, by local infusion, injection, or by an implant that is a porous or non-porous material including membranes and matrices, such as silicone rubber (sialastic) membranes, polymers, fibrous matrices (e.g., tissue @), or collagen matrices. In one embodiment, an effective amount of one or more antibodies of the invention is administered topically to the affected area of the subject to prevent, treat, manage, and/or ameliorate the disorder or symptoms thereof. In another embodiment, an effective amount of one or more antibodies of the invention, in combination with an effective amount of one or more therapies other than an antibody of the invention (e.g., one or more prophylactic or therapeutic agents), is administered topically to the affected area of the subject to prevent, treat, manage, and/or ameliorate the disorder or one or more symptoms thereof.

In another embodiment, the antibody may be delivered in a controlled or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. biomed. Eng. 14: 20; Buchwald et al, 1980, Surgery 88: 507; Saudek et al, 1989, N. Engl. J. Med. 321: 574). In another embodiment, polymeric materials may be used to achieve Controlled or sustained Release of the therapies of the invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres, Boca Raton, Fla (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (ed.), Wiley, New York (1984); Ranger and Peppas, 1983, J.; U.S. patent nos. 5,679,377; U.S. patent nos. 5,916,597; U.S. patent nos. 5,912,015; U.S. patent nos. 5,989,463; U.S. patent nos. 5,128,326; PCT publication Nos. WO 99/15154; and PCT publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly 2-hydroxyethyl methacrylate, poly methyl methacrylate, polyacrylic acid, ethylene-vinyl acetate copolymer, polymethacrylic acid, Polyglycolide (PLG), polyanhydride, poly N-vinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polyethylene glycol, Polylactide (PLA), lactide-glycolide copolymer (PLGA), and polyorthoesters. In certain embodiments, the polymers used in the sustained release formulations are inert, free of leachable impurities, storage stable, sterile, and biodegradable. In another embodiment, a Controlled or sustained Release system can be placed in proximity to a prophylactic or therapeutic target such that only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol.2, pp.115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-. Any technique known to those skilled in the art may be used to produce a sustained release formulation comprising one or more antibodies of the invention. See, for example, U.S. Pat. No. 4,526,938, PCT publication WO91/05548, PCT publication WO96/20698, Ning et al, 1996, "Integrated Radiology of a Human Colon Cancer Using a Sustained-Release Gel," Radiology & Oncology 39: 179. 189, Song et al, 1995, "Integrated medical testing of Long-Circulating Emulsions," Journal of Pharmaceutical Science & Technology 50: 372. 397. Cleek et al, 1997, "degradable polymers for a FGF body for compact for cellular analysis," Pro' Integrated Control. response, 1997, "biological analysis for a biological analysis, 759, incorporated by reference in their entireties, for example," biological analysis "24, for example," biological analysis ".

In particular embodiments, where the composition of the invention is a nucleic acid encoding an antibody, the nucleic acid may be administered in vivo to facilitate expression of the antibody encoded thereby by constructing it as part of a suitable nucleic acid expression vector and administering such that it becomes intracellular, for example using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g. gene gun; Biolistic, Dupont), or coated with lipids or cell surface receptors or transfection agents, or by ligation with homeobox-like peptides known to enter the nucleus (see, for example, Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88: 1864 1868). Alternatively, the nucleic acid may be introduced intracellularly and integrated into the host cell DNA for expression by homologous recombination.

The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In specific embodiments, the compositions are formulated in accordance with conventional procedures as pharmaceutical compositions suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine (lignocamine) to reduce pain at the injection site.

If the compositions of the present invention are to be administered topically, the compositions may be formulated in the form of ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19 th edition, Mack pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms containing a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are generally employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, sterilized if necessary or mixed with adjuvants (e.g., preservatives, stabilizers, humectants, buffers, or salts) for affecting various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations wherein the active ingredient, e.g., in combination with a solid or liquid inert carrier, is packaged in admixture with a pressurized volatile (e.g., a gaseous propellant, e.g., freon) or in a squeeze bottle. Humectants (moisisturisers) or wetting agents may also be added to the pharmaceutical compositions and dosage forms, if desired. Examples of such additional ingredients are well known in the art.

If the method of the invention comprises intranasal administration of the composition, the composition may be formulated in the form of an aerosol, spray, mist or drops. In particular, prophylactic or therapeutic agents for use according to the present invention may conveniently be delivered in the form of an aerosol spray presentation from a pressurised pack or nebuliser, using a suitable propellant (e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) containing a powder mixture of the compound and a suitable powder base such as lactose or starch may be formulated for use in an inhaler or insufflator.

If the methods of the present invention include oral administration, the compositions may be formulated in oral forms such as tablets, capsules, cachets, granular capsules (gelcaps), solutions, suspensions, and the like. Tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, but are not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid formulations may be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous carriers (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens or sorbic acid). The formulations may also contain buffer salts, flavouring agents, colouring agents and sweetening agents, as appropriate. Formulations for oral administration may be suitably formulated for slow, controlled, or sustained release of one or more prophylactic or therapeutic agents.

The methods of the invention may comprise pulmonary administration of a composition formulated with an aerosolizing agent (aerosol agent), for example using an inhaler or nebulizer. See, e.g., U.S. patent nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT publication nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated by reference herein in its entirety. In particular embodiments, the antibodies of the invention, combination therapy, and/or compositions of the invention are administered using Alkermes AIR lung drug delivery technology (Alkermes, inc., Cambridge, Mass.).

The methods of the invention may include administration of compositions formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use. The methods of the invention may additionally include the administration of a composition formulated as a depot (depot) formulation. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

The methods of the invention include the administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Typically, the components of the composition are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate in a sealed container, such as an ampoule or sachet (sachette), which indicates the amount of active agent. When the mode of administration is infusion, the composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the mode of administration is injection, sterile water for injection or saline ampoules may be provided so that the ingredients may be mixed prior to administration.

In particular, the invention also provides one or more antibodies or pharmaceutical compositions of the invention packaged in a sealed container, such as an ampoule or sachet, which indicates the amount of antibody. In one embodiment, one or more antibodies or pharmaceutical compositions of the invention are provided as a dry sterile lyophilized powder or anhydrous concentrate in a sealed container and can be reconstituted (e.g., with water or saline) to a suitable concentration for administration to a subject. In one embodiment, one or more antibodies or pharmaceutical compositions of the invention are provided as a dry sterile lyophilized powder in a sealed container in a unit dose of at least 5 mg, such as at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized antibody or pharmaceutical composition of the invention should be stored at 2-8 ℃ in its original container and the antibody or pharmaceutical composition of the invention should be administered within 1 week after reconstitution, e.g., within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, one or more antibodies or pharmaceutical compositions of the invention are provided in liquid form in a sealed container that indicates the amount and concentration of the antibody. In a further embodiment, the administered composition in liquid form is provided in a sealed container in an amount of at least 0.25 mg/ml, such as at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored in its original vessel at 2-8 ℃.

The antibodies of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration. In one aspect, the antibody will be prepared as an injectable solution comprising 0.1-250 mg/ml of antibody. Injectable solutions may consist of liquid or lyophilized dosage forms in flint or amber vials, ampoules or prefilled syringes. The buffer may be L-histidine (1-50 mM), preferably 5-10 mM, pH 5.0-7.0 (pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used to modify the toxicity of solutions at concentrations of 0-300 mM (150 mM being optimal for liquid dosage forms). For a freeze-dried dosage form, a cryoprotectant may be included, predominantly 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents, mainly 1-10% mannitol (optimally 2-4%) may be included for the freeze-dried dosage form. The stabilizer may be used in liquid and freeze-dried dosage forms, and is mainly 1-50 mM L-methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, and may be included as 0-0.05% polysorbate 80 (most preferably 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. Pharmaceutical compositions comprising the antibodies of the invention prepared as injectable solutions for parenteral administration may further comprise agents that act as adjuvants, such as those for increasing absorption, or dispersion, of the antibodies. A particularly useful adjuvant is hyaluronidase such as Hylenex ® (recombinant human hyaluronidase). The addition of hyaluronidase in injectable solutions improves human bioavailability following parenteral administration, particularly subcutaneous administration. It also allows for larger injection site volumes (i.e., greater than 1 ml) with less pain and discomfort, and minimal incidence of injection site reactions. (see International application publication No. WO 04/078140 and U.S. patent application publication No. US2006104968, both incorporated herein by reference).

The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. The compositions may be in the form of injectable or infusible solutions, for example compositions similar to those used by passive immunization of humans with other antibodies. In one embodiment, the antibody is administered by intravenous infusion or injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions may be prepared by: the required amount of active compound (i.e., a binding protein of the invention such as an antibody) is incorporated with one or a combination of ingredients listed above in a suitable solvent, followed by filter sterilization if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the necessary other ingredients from those enumerated above. In the case of sterile, freeze-dried powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and spray drying of the powder resulting from the previously sterile-filtered solution thereof of the active ingredient plus any additional desired ingredient. The correct fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

The antibodies of the invention can be administered by a variety of methods known in the art. For many therapeutic applications, the route/mode of administration may be subcutaneous injection, intravenous injection, or infusion. As the skilled artisan will recognize, the route and/or mode of administration will vary depending on the desired results. In certain embodiments, the active compound may be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, eds., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the antibodies of the invention may be administered orally, e.g., with an inert diluent or assimilable edible carrier. The antibody (and other ingredients, if desired) may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet of a subject. For oral therapeutic administration, the antibody may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers (wafers), and the like. In order to administer the antibodies of the invention by means other than parenteral administration, it may be necessary to coat the antibody with a material, or to co-administer the antibody with a material, to prevent its inactivation.

Supplementary active compounds may also be incorporated into the compositions. In certain embodiments, the antibodies of the invention are co-formulated and/or co-administered with one or more additional therapeutic agents for treating the disorders or diseases described herein. For example, an anti-a β (20-42) globulomer antibody of the invention may be coformulated with and/or coadministered with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to other soluble antigens or to cell surface molecules). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding the potential toxicity or complications associated with each monotherapy.

In certain embodiments, the antibodies of the invention are linked to a half-life extending carrier known in the art. Such carriers include, but are not limited to, Fc domains, polyethylene glycols, and dextrans. Such vectors are described, for example, in U.S. application serial No. 09/428,082 and published PCT application No. WO 99/25044, which are incorporated herein by reference for any purpose.

In particular embodiments, a nucleic acid sequence comprising a nucleotide sequence encoding an antibody of the invention is administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof via gene therapy. Gene therapy refers to therapy by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded antibody of the invention that mediates a prophylactic or therapeutic effect.

Any method available in the art for gene therapy may be used according to the present invention. For a general review of gene therapy methods, see Golddspiel et al, 1993, Clinical Pharmacy 12: 488-505; wu and Wu, 1991, Biotherapy 3: 87-95; tolstoshiev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. biochem. 62: 191-217; year 1993, month 5, TIBTECH 11 (5): 155-. Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). A detailed description of various gene therapy methods is disclosed in US20050042664, which is incorporated herein by reference.

The antibodies of the invention may be used alone or in combination to treat diseases such as alzheimer's disease, down's syndrome, dementia, parkinson's disease or any other disease or condition associated with the accumulation of beta amyloid in the brain. The antibodies of the invention may be used to treat "conformational diseases". Such diseases arise from secondary to tertiary structural changes within the constituent proteins with subsequent aggregation of the altered proteins (Hayden et al, JOP. J Panceas 2005; 6 (4): 287-302). In particular, the antibodies of the invention may be used to treat one or more of the following conformational diseases: alpha 1-antitrypsin deficiency, C1-inhibitor deficiency angioedema, antithrombin deficiency thromboembolic disease, kuru, Creutzfeldt-Jakob disease/scrapie, bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker disease, familial fatal insomnia, Huntington's disease, spinocerebellar ataxia, Machado-Joseph atrophy, dentatorubral pallidoluysian, frontotemporal dementia, sickle cell anemia, labile hemoglobin inclusion body hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic AL amyloidosis, nodular AL amyloidosis, systemic AA amyloidosis, prostate amyloidosis, hemodialysis amyloidosis, hereditary (iceland) cerebrovascular disease, Huntington's disease, familial visceral amyloidosis, familial visceral polyneuropathy, chronic disorders of the brain, chronic myelogenous leukemia, chronic myelogenous, familial visceral amyloidosis, senile systemic amyloidosis, familial amyloid neuropathy, familial cardiac amyloidosis, Alzheimer's disease, Down's syndrome, medullary thyroid carcinoma, and type 2 diabetes (T2 DM). Preferably, the antibodies of the invention may be used for the treatment of amyloid proteins such as alzheimer's disease and down's syndrome.

It is understood that the antibodies of the invention can be used alone or in combination with one or more additional agents, such as therapeutic agents (e.g., small molecules or biologies), which are selected by the skilled artisan according to their intended purpose. For example, the additional therapeutic agent may be a "cognitive enhancer drug," which is a drug that improves impaired cognitive ability (i.e., thinking, learning, and memory) of the human brain. Cognitive enhancing drugs act by altering the availability of neurochemicals (e.g., neurotransmitters, enzymes and hormones), improving oxygen supply, stimulating nerve growth or inhibiting nerve damage. Examples of cognition enhancing drugs include compounds that increase the activity of acetylcholine such as, but not limited to, acetylcholine receptor agonists (e.g., nicotinic alpha-7 receptor agonists or allosteric modulators, alpha 4 beta 2 nicotinic receptor agonists or allosteric modulators), acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, and galantamine), butyrylcholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine), activity-dependent neuroprotective protein (ADNP) agonists, serotonin 5-HT 1A receptor agonists (e.g., zalioden), 5-HT ₄ Receptor agonists, 5-HT ₆ Receptor antagonists, serotonin 1A receptor antagonists, histamine H ₃ Receptor antagonists, calpain inhibitors, Vascular Endothelial Growth Factor (VEGF) proteins or agonists, nutritional supplementsGrowth factors, anti-apoptotic compounds, AMPA-type glutamate receptor activators, L-or N-type calcium channel blockers or modulators, potassium channel blockers, Hypoxia Inducible Factor (HIF) activators, HIF prolyl 4-hydroxylase inhibitors, anti-inflammatory agents, inhibitors of amyloid a β peptide or amyloid plaques, tau hyperphosphorylation inhibitors, phosphodiesterase 5 inhibitors (e.g., tadalafil, sildenafil), phosphodiesterase 4 inhibitors, monoamine oxidase inhibitors, or pharmaceutically acceptable salts thereof. Specific examples of such cognitive enhancing drugs include, but are not limited to, cholinesterase inhibitors such as donepezil (Aricept) ^® ) Rivastigmine (Exelon) ^® ) Galantamine (remininyl) ^® ) N-methyl-D-aspartate antagonists such as memantine (Namenda) ^® ). The at least one cognition enhancement drug may be administered simultaneously with or sequentially (and in any order) with an antibody of the invention, including those agents presently recognized or recognized in the future as useful for treating a disease or condition to be treated by an antibody of the invention. In addition, it is contemplated that the combinations described herein may have additional or synergistic effects when used in the above-described treatments. The additional agent may also be an agent that imparts a beneficial attribute to the therapeutic composition, such as an agent that affects the viscosity of the composition.

It is further understood that combinations to be included within the invention are those useful for their intended purposes. The reagents described above are for illustrative purposes and are not intended to be limiting. A combination as part of the invention may comprise an antibody of the invention and at least one additional agent selected from the group consisting of. The combination may also include more than one additional agent, e.g., two or three additional agents, if the combination is such that the resulting composition can perform its intended function.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody portion can be determined by one of skill in the art and will vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effect is greater than any toxic or deleterious effect of the antibody. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, because a prophylactic dose is used in a subject prior to or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The dosage regimen may be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions are particularly advantageous for ease of administration and consistent dosage forms. As used herein, unit dosage form refers to physically discrete units suitable as a single dose for a mammalian subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The detailed description of the unit dosage forms of the invention is indicated by and directly dependent on the following: (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of formulating such active compounds for use in the treatment of sensitivity in an individual.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody of the invention is 0.1-20 mg/kg, e.g., 1-10 mg/kg. It should be noted that the dosage value may vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods of the invention described herein may be made without departing from the scope of the invention or the embodiments disclosed herein using suitable equivalents. While the present invention has now been described in detail, the invention will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to be limiting of the invention.

Examples

Example 1: preparation of globulomers

a) A β (1-42) globulomer:

a β (1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended at 6 mg/ml in 100%1,1,1,3,3, 3-hexafluoro-2-propanol (HFIP) and incubated at 37 ℃ for 1.5 hours with shaking for complete dissolution. HFIP acts as a hydrogen bond disrupter and serves to eliminate pre-existing structural heterogeneity in a β peptides. HFIP was removed by evaporation in SpeedVac and a β (1-42) was resuspended in dimethyl sulfoxide at a concentration of 5 mM and sonicated for 20 seconds. HFIP-pretreated A β (1-42) was dissolved in Phosphate Buffered Saline (PBS) (20 mM NaH) ₂ PO ₄ 140 mM NaCl, pH 7.4) to 400 μ M and 1/10 volumes of 2% Sodium Dodecyl Sulfate (SDS) (H) were added ₂ O solution) (final concentration of 0.2% SDS). Incubation at 37 ℃ for 6 hours resulted in an intermediate product of the 16/20-kDa A.beta. (1-42) globulomer (simple form for the globulomer). By using three volumes H ₂ Further dilution with O and incubation at 37 ℃ for 18 hours produced 38/48-kDa Abeta (1-42) globulomers. After centrifugation at 3000 g for 20 min, the sample was concentrated by ultrafiltration (30-kDa cut-off) against 5mM NaH ₂ PO ₄ 35mM NaCl, pH 7.4, dialyzed at 10,000gCentrifugation was carried out for 10 minutes and the supernatant containing 38/48-kDa A.beta. (1-42) globulomer was removed. As an alternative to dialysis, the 38/48-kDa A.beta. (1-42) globulomer can also be precipitated by a nine-fold excess (v/v) of ice-cold methanol/acetic acid solution (33% methanol, 4% acetic acid) at 4 ℃ for 1 hour. 38/48-kDa Abeta (1-42) spheroids were subsequently pelleted (at 16200 g for 10 min) and resuspended in 5mM NaH ₂ PO ₄ 35mM NaCl, pH 7.4, and the pH was adjusted to 7.4.

b) A β (20-42) globulomer:

1.59 ml of A β (1-42) globulomer preparation prepared according to example 1a was mixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 μ l of a 1 mg/ml aqueous solution of thermolysin (Roche). The reaction mixture was stirred at RT for 20 h. Subsequently, 80 μ l of 100 mM EDTA, an aqueous solution of pH 7.4 was added, and the mixture was additionally adjusted to a SDS content of 0.01% with 400 μ l of a 1% strength SDS solution. The reaction mixture was concentrated to about 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate was mixed with 9 ml of buffer (50 mM MES/NaOH, 0.02% SDS, pH 7.4) and concentrated again to 1 ml. The concentrate was dialyzed against 1 l buffer (5 mM sodium phosphate, 35mM NaCl) at 6 ℃ for 16 hours in a dialysis tube. The dialysate was adjusted to 0.1% SDS content with an aqueous solution of 2% strength SDS. Samples were run at 10,000 deg.C gCentrifugation was carried out for 10 minutes, and the supernatant of A β (20-42) globulomer was removed.

c) A β (12-42) globulomer:

2 ml of A β (1-42) globulomer preparation prepared according to example 1a were mixed with 38 ml of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and 150 μ l of 1 mg/ml aqueous GluC intracellular protease (Roche). The reaction mixture was stirred at RT for 6 hours and subsequently a further 150. mu.l of 1 mg/ml aqueous GluC intracellular protease (Roche) solution was added. The reaction mixture was stirred at RT for an additional 16 hours, followed by the addition of 8 μ l of 5M diff solution. The reaction mixture was concentrated to about 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate was mixed with 9 ml of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and concentrated again to 1 ml. The concentrate was dialyzed against 1 l buffer (5 mM sodium phosphate, 35 mM NaCl) at 6 ℃ for 16 hours in a dialysis tube. The dialysate was adjusted to 0.1% SDS content with an aqueous solution of 1% strength SDS. Samples were run at 10,000 deg.CgCentrifugation was carried out for 10 minutes, and the supernatant of A β (12-42) globulomer was removed.

d) Crosslinked a β (1-42) globulomer:

a.beta. (1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended at 6 mg/ml in 100%1,1,1,3,3, 3-hexafluoro -2-propanol (HFIP) and incubation at 37 ℃ for 1.5 hours with shaking for complete dissolution. HFIP acts as a hydrogen bond disrupter and serves to eliminate pre-existing structural heterogeneity in a β peptides. HFIP was removed by speedVac evaporation, and A β (12-42) globulomer A β (1-42) was resuspended in dimethyl sulfoxide at a concentration of 5 mM and sonicated for 20 seconds. HFIP-pretreated A β (1-42) was dissolved in PBS (20 mM NaH) ₂ PO ₄ 140 mM NaCl, pH 7.4) to 400 μ M and 1/10 volumes of 2% SDS (aqueous solution) (final concentration of 0.2% SDS) were added. Incubation at 37 ℃ for 6 hours resulted in an intermediate product of 16/20-kDa A.beta. (1-42) globulomer (for the simple form of globulomer oligomer). By further dilution with 3 volumes of water and incubation at 37 ℃ for 18 hours, 38/48-kDa A.beta. (1-42) globulomers were generated. Crosslinking of the 38/48-kDa A.beta. (1-42) globulomer was now performed by incubation with 1 mM glutaraldehyde at 21 ℃ for 2 hours at room temperature followed by treatment with ethanolamine (5 mM) for 30 minutes at room temperature.

Example 2: generation, isolation and characterization of humanized anti-A β (20-42) globulomer antibodies

Example 2.1: selection of human antibody frameworks

The human antibody frameworks are selected based on similarity of canonical structures and amino acid sequence homology of human antibodies. Further, when identifying suitable acceptor VL and VH framework sequences based on amino acid sequence homology of human VH and vk germline sequences, it is contemplated to retain the amino acid residues that support the loop structure and VH/VL interface and to retain the amino acid residues of the Vernier zone. In addition, based on the predicted affinities of overlapping peptides for various MHC class I and/or MHC class II alleles, the immunogenicity of VH and VL sequences resulting from grafting the 4D10 CDRs into potentially suitable acceptor VL and VH framework sequences was evaluated in silico. The VH and VL are adapted to the respective VH or VL family consensus to further minimize potential immunogenicity. Selected back mutations to murine amino acid residues were performed to retain the amino acids that support the loop structure and VH/VL interface. The frequency of these back mutations in the corresponding repertoire of naturally occurring human VH or VL sequences with the respective VH or VL germline genes is determined by amino acid sequence alignment. Potential N-linked glycosylation sites (NXS or NXT, where X is any amino acid except P) were examined in the VH and VL sequences resulting from the considerations described above.

Example 2.2: humanization of murine anti-A beta (20-42) globulomer antibodies

4D10hum _ VH.1z (SEQ ID NO: 4): heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human VH3-53 and JH6 sequences.

4D10hum _ VH.1 (SEQ ID NO: 5): the heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into an acceptor framework of human VH3-53 and JH6 sequences comprising the VH3 consensus change I12V.

4D10hum _ VH.1a (SEQ ID NO: 6): heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into the acceptor framework of human VH3-53 and JH6 sequences comprising VH3 consensus change I12V, and framework back-mutations a24V, V29L, V48L, S49G, F67L, R71K, N76S, and L78V.

4D10hum _ VH.1b (SEQ ID NO: 7): heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human VH3-53 and JH6 sequences, which comprise back mutations V29L and R71K.

4D10hum _ VH.2z (SEQ ID NO: 8): heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human VH4-59 and JH6 sequences.

4D10hum _ VH.2 (SEQ ID NO: 9): heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human VH4-59 and JH6 sequences, which contain Q1E changes to prevent N-terminal pyroglutamate formation.

4D10hum _ VH.2a (SEQ ID NO: 10): the heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into the acceptor framework of human VH4-59 and JH6 sequences, which contains a Q1E change to prevent N-terminal pyroglutamate formation, and framework back-mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S, and F78V.

4D10hum _ VH.2b (SEQ ID NO: 11): the heavy chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human VH4-59 and JH6 sequences, which contain Q1E changes to prevent N-terminal pyroglutamate formation, and framework reversion mutations G27F, I29L and V71K.

4D10hum _ Vk.1 z (SEQ ID NO: 12): the light chain CDR sequences from the murine anti a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human vka 17/2-30 and jk 2 sequences.

4D10hum _ V.kappa.1 (SEQ ID NO: 13): the light chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human vka 17/2-30 and jk 2 sequences, which comprise the V κ 2 consensus changes S7T, L15P, Q37L, R39K and R45Q.

4D10hum _ V.kappa.1 a (SEQ ID NO: 14): the light chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into the acceptor framework of human vka 17/2-30 and jk 2 sequences, which comprises V κ 2 consensus changes S7T, L15P, Q37L, R39K and R45Q, as well as the framework back mutation F36L affecting the VL/VH interface.

4D10hum _ Vk.1 b (SEQ ID NO: 15): the light chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into acceptor frameworks of human vka 17/2-30 and jk 2 sequences, which contained vk 2 consensus changes S7T and Q37L.

4D10hum _ Vk.1 c (SEQ ID NO: 16): the light chain CDR sequences from murine anti-a β (20-42) globulomer antibody 4D10 described in table 4 were grafted into the acceptor framework of human vka 17/2-30 and jk 2 sequences, which comprises vk 2 consensus changes S7T, Q37L, and R39K.

Some of the VH and vk back mutations, consensus changes, or Q1E mutations in 4D10hum _ vh.2, 4D10hum _ vh.2a, or 4D10hum _ vh.2b may be removed during subsequent affinity maturation.

Example 2.3: construction of humanized antibody

The humanized antibodies constructed in silico as described above were newly constructed using oligonucleotides. For each variable region cDNA, 6 oligonucleotides of 60-80 nucleotides each were designed to overlap each other by 20 nucleotides at the 5 'and/or 3' end of each oligonucleotide. In the annealing reaction, all 6 oligonucleotides were combined, boiled and annealed in the presence of dNTPs. DNA polymerase I, a large (Klenow) fragment (New England Biolabs # M0210, Beverley, MA.) was then added to fill in the approximately 40 bp gap between overlapping oligonucleotides. PCR will then be performed using the two outermost primers containing overhang sequences complementary to the multiple cloning sites in the modified pBOS vector to amplify the entire variable region gene (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol.18, No. 17)). The PCR products derived from each cDNA assembly were separated on an agarose gel and the band corresponding to the predicted variable region cDNA size was excised and purified. The variable heavy region was inserted in-frame by homologous recombination in bacteria onto a cDNA fragment encoding the constant region of human IgG1 containing 2 hinge region amino acid mutations. These mutations are a leucine to alanine alteration at position 234 (EU numbering) and a leucine to alanine alteration at position 235 (Lund et al, 1991, J. Immunol., 147: 2657). The variable light chain region was inserted in frame with the human kappa constant region by homologous recombination. Separating bacterial colonies and extracting plasmid DNA; the cDNA insert will be sequenced in its entirety. The correct humanized heavy and light chains corresponding to each antibody were co-transfected into COS cells to transiently produce full-length humanized anti-a β globulomer antibodies. Cell supernatants containing recombinant chimeric antibodies were purified by protein a sepharose chromatography and bound antibodies were eluted by addition of acid buffer. The antibodies were neutralized and dialyzed into PBS. (D.Moechars et al J Biol Chem 274: 6483-6492 (1999); Ausubel, F.M. et al eds., Short Protocols In Molecular Biology (4 th edition 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X); Lu and Weiner eds., Cloning and Expression Vectors for Gene Function Analysis (2001) Biotechnology Press. Westborough, MA. page 298 (ISBN 1-881299-21-X); Kontennrma and Dubel eds., Cloning Engineering (2001) Spring-Verlag. New York. No. 790 (ISBN 3-540 Man 354-5); Ombr R.W. software Engineering (2001) page 41J.1982. edition J.Biodistribution J.1982; clone J.1983: Aust J.12: Microelectronics J.1982; Aust. 1984: Microbin edition), NY. volumes 1-3 (ISBN 0-87969-309-6); winnacker, e.l. From Genes To Clones: introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts) page 634 (ISBN 0-89573-614-4); all of which are incorporated by reference in their entirety).

While a number of embodiments and features have been described above, it will be appreciated by those skilled in the art that modifications and variations may be made to the described embodiments and features without departing from the disclosure of the invention as defined in the appended claims.

Example 2: 4: expression and purification of humanized antibodies in HEK293 cells

Preparation of a heavy chain encoding the antibody as set forth in SEQ ID NO 46 as described in example 2.3; a DNA construct of an antibody heavy chain as set forth in SEQ ID NO. 47, and an antibody light chain construct encoding a polypeptide as set forth in SEQ ID NO. 48. After confirmation of the DNA by sequencing, all heavy and light chain DNA constructs were amplified in e.coli and the DNA was purified using Qiagen Endo Free Plasmid Maxi Prep (catalog #12362, Qiagen) according to the manufacturer's protocol.

For the expression of monoclonal antibody 4D10hum #1, HEK293 (EBNA) cells were transiently co-transfected with plasmids encoding the heavy chain shown in SEQ ID NO:46 and the light chain shown in SEQ ID NO: 48. For the expression of monoclonal antibody 4D10hum #2, HEK293 (EBNA) cells were transiently co-transfected with plasmids encoding the heavy chain shown in SEQ ID NO:47 and the light chain shown in SEQ ID NO: 48. HEK293 (EBNA) cells were plated on a 0.5 l scale in CO prior to transfection ₂ Incubator (8% CO) ₂ Propagation in Freestyle 293 medium (Invitrogen, Carlsbad CA) at 125 rpm, 37 ℃ in shaken flasks. When the cell culture reached 1X 10 ⁶ Density of cells/ml, cells were transfected by addition of transfection complex. Transfection complexes were prepared by first mixing 150 μ g of plasmid encoding the light chain, 100 μ g of plasmid encoding the heavy chain and 25 ml of Freestyle medium, followed by addition of 500 μ l PEI solution (1 mg/ml (pH 7.0) linear 25 kDa polyethyleneimine, Polysciences catalog # 23966). The transfection complexes were mixed by inversion and incubated for 15 minutes at room temperature before addition to the cell culture. After transfection, the culture was continued in CO ₂ Incubator (8% CO) ₂ 125 rpm, 37 ℃ C.). Twenty-four hours after transfection, the medium was supplemented with 50 ml of 5% Tryptone N1 solution (Organo Technie, La courneu France catalog # 19553). Six days after transfection, cells were pelleted by centrifugation (16,000 g, 30 min), the supernatant containing the expressed antibody was sterile filtered (0.2 μm PES filter) and placed at 4 ℃ until the start of the purification step. The expressed antibodies were purified from the supernatant by protein a sepharose affinity chromatography using Thermo Scientific reagents and according to the protocol of the manufacturer's instructions. The protein eluate was dialyzed against PBS (pH 7). The purified 4D10hum antibody was quantified in a 280 nm spectrophotometer and analyzed by mass spectrometry and Size Exclusion Chromatography (SEC).

Example 2: 5: affinity assay for humanized antibodies

The purified humanized antibodies 4D10hum #1 and 4D10hum #2 were evaluated for interaction with a β (20-42) globulomer by Surface Plasmon Resonance (SPR) analysis using a BIAcore apparatus. Goat anti-human IgG Fc (10,000 RU) was immobilized directly onto CM5 sensor chips by an amine coupling procedure according to the manufacturer's instructions (BIAcore). The respective 4D10hum antibodies were captured onto goat anti-human IgG Fc coated chip surfaces by injecting 5.0 μ l of a 1 μ g/ml 4D10hum antibody solution at a flow rate of 10-15 μ l/min. Soluble A β (20-42) globulomer and sensor chip were examined by injecting globulomer solution (concentration range: 20-0.3125 nM) at a flow rate of 50 μ l/minInteraction of the 4D10hum antibody above. The on-rate was monitored for 5.0 minutes and the off-rate was monitored for 10 minutes. From the resulting sensorgram, the binding rate constant (k) was determined using the manufacturer's software and instructions _on ) Dissociation rate constant (k) _off ) And equilibrium dissociation constant (K) _D ). The kinetic and equilibrium constants determined from three different preparations of 4D10hum #1 and two different preparations of 4D10hum #2 are summarized in table 7. Table 7 also shows affinity data for antibodies #3, #4 and #5 with chimeric and humanized chains. The heavy chains of antibodies #4 and #5 were as in 4D10hum #1 or #2, and the light chain was a chimera of m4D10 VL (SEQ ID NO: 24) and the human Ig kappa constant region (SEQ ID NO: 27). The light chain of antibody #3 was as in 4D10hum #1 and #2, and the heavy chain was a chimera of m4D10 VH (SEQ ID NO 23) and human Ig γ -1 constant region (SEQ ID NO: 25).

Table 7: affinity of 4d10hum antibody for A β (20-42) globulomer

TABLE 7 continuation

Example 2.6: antibody selective analysis via dot blot

To characterize the selectivity of monoclonal anti-A β (20-42) globulomer antibodies, they were tested for binding to different A β forms. To this end, serial dilutions of individual A β (1-42) forms in PBS supplemented with 0.2 mg/ml BSA in the range of 100 pmol/μ l-0.00001 pmol/μ l were prepared. 1 μ l of each dilution was spotted onto a nitrocellulose membrane. Detection was performed by incubation with the corresponding antibody (0.2 μ g/ml) followed by immunostaining with peroxidase-conjugated anti-human IgG and staining reagent BM Blue POD (Roche).

A β standard for dot blotting:

1. abeta (1-42) globulomer

A β (1-42) globulomers were prepared as described in example 1a (buffer exchange, by dialysis).

2. Abeta (20-42) globulomer

A β (20-42) globulomer was prepared as described in example 1 b.

3. Abeta (1-40) monomer, 0.1% NaOH

2.5 mg of A β (1-40) (Bachem Inc., Cat. H-1368) was dissolved in 0.5 ml of 0.1% NaOH in H ₂ O solution (freshly prepared) (= 5 mg/ml), and immediately shaken at room temperature for 30 seconds to obtain a clear solution. The samples were stored at-20 ℃ until use.

4. Abeta (1-42) monomer, 0.1% NaOH

2.5 mg of Ass (1-42) (Bachem Inc., Cat. H-1368) was dissolved in 0.5 ml of 0.1% NaOH in H ₂ O solution (freshly prepared) (= 5 mg/ml), and immediately shaken at room temperature for 30 seconds to obtain a clear solution. The samples were stored at-20 ℃ until use.

5. Abeta (1-42) filament

1 mg of A β (1-42) (Bachem Inc. catalog number: H-1368) was dissolved in 500 μ l of aqueous 0.1% NH ₄ OH (Eppendorf tube) and stirred at room temperature for 1 minute. 20 mM NaH at 300. mu.l ₂ PO ₄ (ii) a 100 μ l of this freshly prepared A β (1-42) solution was neutralized with 140 mM NaCl, pH 7.4. The pH was adjusted to pH 7.4 with 1% HCl. Samples were incubated at 37 ℃ for 24 hours and centrifuged (at 10000g for 10 minutes). The supernatant was discarded and vortexed for 1 minute with 400 μ l of 20 mM NaH ₂ PO ₄ (ii) a 140 mM NaCl, pH 7.4 resuspend the fibril pellet.

6. sAPPα

By Sigma (cat # S9564; at 20 mM NaH) ₂ PO ₄ (ii) a 140 mM NaCl; 25 mug in pH 7.4). With 20 mM NaH ₂ PO ₄ 140 mM NaCl pH 7.4, 0.2 mg/ml BSA sAPP α was diluted to 0.1 mg/ml (= 1 pmol/. mu.l).

7. Abeta (12-42) globulomer

A β (12-42) globulomer was prepared as described in example 1 c.

Materials for dot blotting:

a.beta.standard (see 1. to 7. above) at 20 mM NaH ₂ PO ₄ 140 mM NaCl, pH 7.4 + 0.2 mg/ml BSA to obtain the following concentrations: 100 pmol/μ l, 10 pmol/μ l, 1 pmol/μ l, 0.1 pmol/μ l, 0.01 pmol/μ l, 0.001 pmol/μ l, 0.0001 pmol/μ l, and 0.00001 pmol/μ l.

Cellulose nitrate: trans-dot transfer medium, pure nitrocellulose membrane (0.2 μm); BIO-RAD

anti-human-POD: catalog number: 109-

Detection reagent: BM Blue POD Substrate, sediment, catalog No.: 11442066001 (Roche)

Bovine serum albumin, (BSA): catalog number: 11926 (Serva)

Blocking reagent: TBS solution of 5% Low fat milk

Buffer solution:

TBS: 25 mM Tris/HCl buffer pH 7.5 + 150 mM NaCl

TTBS: 25 mM Tris/HCl-buffer pH 7.5 + 150 mM NaCl + 0.05% Tween 20

PBS + 0.2 mg/ml BSA: 20 mM NaH2PO4 buffer pH 7.4 + 140 mM NaCl + 0.2 mg/ml BSA

Antibody solution I: 0.2 mug/ml antibody in 20 ml TBS solution with 1% low fat milk

Antibody: humanized monoclonal anti-a β antibody 4D10hum # 1; 4.7 mg/ml OD 280 nm; storing at-80 deg.C

Antibody solution II: anti-human-POD was diluted 1:5000 in 1% low fat milk in TBS.

Dot blot procedure:

1) different a β standards (obtained by serial dilution) at 8 concentrations of 1 μ l each were spotted on a nitrocellulose membrane at a distance of about 1 cm from each other.

2) The spots of the a β standard were allowed to dry on nitrocellulose membrane at Room Temperature (RT) for at least 10 minutes in air. (= dot blot).

3) And (3) sealing:

dot blots were incubated with 30 ml of 5% low fat milk in TBS for 1.5 hours at RT.

4) Washing:

the blocking solution was discarded and the dot blot was incubated with 20 ml of TTBS for 10 minutes at RT with shaking.

5) Antibody solution I:

the wash buffer was discarded and the dot blot was incubated with antibody solution I for 2 hours at RT.

6) Washing:

antibody solution I was discarded and the dot blot was incubated with 20 ml TTBS for 10 minutes at RT with shaking. The wash solution was discarded and the dot blot was incubated with 20 ml of TTBS for 10 minutes at RT with shaking. The wash solution was discarded and the dot blot was incubated with 20 ml TBS for 10 minutes at RT with shaking.

7: antibody solution II:

the wash buffer was discarded and the dot blot was incubated with antibody solution II for 1 hour at RT.

8) Washing:

antibody solution II was discarded and the dot blot was incubated with 20 ml TTBS for 10 minutes at RT with shaking. The wash was discarded and the dot blot was incubated with 20 ml of TTBS for 10 minutes at RT with shaking. The wash solution was discarded and the dot blot was incubated with 20 ml TBS for 10 minutes at RT with shaking.

9) Color development:

the wash was discarded. Dot blots were developed with 7.5 ml BM Blue POD Substrate for 10 min. By using H ₂ O wash the dot blot strongly to stop the color development. Quantitative evaluation was done based on densitometric analysis of spot intensities (GS 800 densitometer (BioRad) and software package Quantity one, version 4.5.0 (BioRad)). Only spots with a relative density of 20% greater than the relative density of the last optically unambiguously identified A β (20-42) globulomer spot were evaluated. This threshold was determined independently for each dot blot. The calculated values indicate the relationship between the recognition of the A β (20-42) globulomer and the respective A β form for a given antibody.

Dot blot analysis was performed with humanized monoclonal anti-a β antibody 4D10hum # 1. Individual a β forms were applied as serial dilutions and incubated with the respective antibodies for immunoreactions (1 = a β (1-42) globulomer; 2 = a β (20-42) globulomer; 3 = a β (1-40) monomer, 0.1% NaOH; 4 = a β (1-42) monomer, 0.1% NaOH; 5 = a β (1-42) filament preparation; 6 = sappa α (Sigma); first spot: 1 pmol). The results are summarized in table 8.

Table 8: dot blot quantitative data

Antigens	Antibody: 4D10hum #1
		Abeta (1-42) globulomer	>10000
Abeta (20-42) globulomer	1
		0.1% NaOH solution of Abeta (1-40) monomer	72000
0.1% NaOH solution of A beta (1-42) monomer	72000
		Abeta (1-42) filament	>10000
sAPPα	>100
		Abeta (12-42) globulomer	11

Example 3: determination of platelet factor 4 cross-reactivity

Example 3.1: determination of cross-reactivity with platelet factor 4 in cynomolgus monkey plasma via sandwich ELISA

List of reagents:

f96 Cert, Maxisorp NUNC-Immuno Plate catalog No. 439454

Binding antibody in experiment E1:

-humanized monoclonal anti a β antibody 4D10hum # 1; 2.36 mg/ml OD 280 nm; storing at-80 deg.C

-humanized monoclonal anti a β antibody 4D10hum # 2; 1.74mg/ml OD 280 nm; storing at-80 deg.C

Human/mouse chimeric anti- Α β monoclonal antibody clone h1G5 wild-type Fc-box (chim h1G5 wt); 0.99 mg/ml OD 280 nm; stored at-80 deg.C (used as a positive control)

Affinity purified human polyclonal antibody hIgG1 (chemicon (millipore), catalogue # AG 502); 1.00 mg/ml OD 280 nm; stored at-80 deg.C (used as a negative control)

Binding antibody in reference experiment R1:

-an anti-HPF 4 monoclonal antibody; 4.2 mg/ml OD 280 nm; abcam catalog No. ab 49735; stored at-30 deg.C (used as a positive control)

-anti-a β monoclonal antibody clone m1G 5; 1.70 mg/ml OD 280 nm; storing at-80 deg.C

-anti-a β monoclonal antibody clone m4D 10; 8.60 mg/ml OD 280 nm; storing at-80 deg.C

Monoclonal antibody clone mIgG2 a; 7.89 mg/ml OD 280 nm; stored at-80 deg.C (used as a negative control)

Coating buffer solution: 100 mM sodium bicarbonate; pH 9.6

Blocking reagents for ELISA; roche Diagnostics GmbH catalog number: 1112589

PBST buffer: 20 mM NaH ₂ PO ₄ ；140 mM NaCl；0.05% Tween 20；pH 7.4

PBST + 0.5% BSA buffer: 20 mM NaH ₂ PO ₄ (ii) a 140 mM NaCl; 0.05% Tween 20; pH 7.4 + 0.5% BSA; serva directory number 11926

Cynomolgus monkey plasma: cynomolgus EDTA plasma pools from 13 different donors; storing at-30 deg.C

Trypsin inhibitor: sigma catalogue number T7902

A first antibody: pRAb-HPF 4; 0.5 mg/ml; abcam Cat No. ab9561

Labeling reagent: an anti-rabbit-POD conjugate; jackson ImmunoResearch ltd. catalog No.: 111-036-045

Dyeing liquid: a DMSO solution of 42 mM TMB (Roche Diagnostics GmbH Cat number: 92817060); 3% H ₂ O ₂ An aqueous solution of (a); 100 mM sodium acetate, pH 4.9

Stopping liquid: 2M sulfuric acid

Methods used in the preparation of reagents:

binding of antibody:

the bound antibody was diluted to 10 μ g/ml in coating buffer.

Sealing liquid:

blocking reagents were dissolved in 100 ml water to prepare blocking stock solutions, and 10 ml aliquots were stored at-20 ℃. 3ml of the blocking stock was diluted with 27 ml of water for blocking each plate.

Preparation of plasma stock of cynomolgus (cynomolgus) (Macaca fascicularis):

2 ml cynomolgus monkey plasma pools at 10,000gCentrifuge for 10 minutes. 1.58 ml of supernatant was removed and diluted with 3.42 ml PBST + 0.5% BSA buffer (= 1:3.16 dilution). Subsequent addition of 50. mu.l of 10 mg/ml trypsin inhibitor H ₂ And (4) O solution. After incubation for 10 minutes at room temperature, the samples were filtered through a 0.22 μm filter (Millipore catalog number SLGS 0250S).

Dilution series of cynomolgus monkey plasma stock:

primary antibody solution:

primary antibodies were diluted to 1 μ g/ml in PBST + 0.5% BSA buffer. The dilution factor is 1: 500. The antibody solution was used immediately.

Labeling reagent:

the anti-rabbit-POD conjugate lyophilisate was reconstituted in 0.5 ml water. 500 μ l of glycerol was added and 100 μ l aliquots were stored at-20 ℃ for further use. The concentrated labeling reagent was diluted in PBST buffer. The dilution factor is 1: 10000. The reagent was used immediately.

TMB solution:

20 ml of 100 mM sodium acetate, pH 4.9 was mixed with 200 μ l of TMB stock solution and 29.5 μ l of 3% peroxide solution. The solution was used immediately.

Standard plate set-up for experiment E1. Dilution of cynomolgus monkey plasma. It should be noted that each sample was run in duplicate.

The standard plate set for reference experiment R1. Dilution of cynomolgus plasma. It should be noted that each sample was run in duplicate.

The procedure used was:

1. 100 μ l of binding antibody solution/well was applied and incubated overnight at 4 ℃.

2. The antibody solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

3. 265 μ l blocking solution/well was added and incubated at room temperature for 1.5 hours.

4. The blocking solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

5. After serial preparation of dilutions of cynomolgus monkey plasma, 100 μ l/well of these dilutions were applied to the plates. The plates were incubated at room temperature for 2 hours.

6. Cynomolgus plasma dilutions were discarded and wells were washed three times with 250 μ l PBST buffer.

7. 100 μ l primary antibody solution/well was added and incubated at room temperature for 1 hour.

8. The primary antibody solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

9. 200 μ l of marker solution/well was added and incubated for 1 hour at room temperature.

10. The labeling solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

11. 100 μ l of TMB solution was added to each well.

12. The plate color was monitored during development (5-15 minutes at ambient temperature) and when the appropriate color had developed, the reaction was stopped by adding 50 μ l/well of stop solution.

13. The absorbance was read at 450 nm.

And (3) data analysis:

plasma dilution factor (X value) was logarithmically converted using the following equation: x = log (X). Data were plotted using X values expressed on the X axis as logarithmic transformation of plasma dilution (1: X). The OD of the respective PBST blanks in row H was subtracted from the values of the plasma dilution series for each column in rows A-G _450nm The value is obtained. Plotting the resulting background corrected OD on the Y-axis _450nm The value is obtained. The dilution effect curve was calculated from these data points by curve fitting using the nonlinear regression "four parameter logistic equation" with the "least squares (normal) fit" fitting method (which is equal to the fitting method "sigmoidal dose response (variable slope)") using the data analysis Software package GraphPadPrism (version 5.03; GraphPad Software Inc.). Curve fitting is performed for the sole purpose of data visualization, but not as a basis for any further calculations, i.e. area under curve calculations. Data based on non-curve fitting in the measurement range (final plasma dilution of about 1:3.16 to about 1: 3160), X-value and OD of logarithmic conversion _450nm Value under the measurement CurveArea (AUC, or total peak area). The following calculations were set up for use in the data analysis Software package GraphPadPrism (version 5.03; GraphPad Software Inc.):

baseline set Y = 0.0.

-minimum peak height: peaks less than 10% of the distance from the minimum to the maximum Y are disregarded.

-peak direction: by definition, all peaks must exceed the baseline

For each individual antibody, the PF4 screening factor was calculated using a commercially available anti-HPF 4 antibody (Abcam catalog number: ab 49735) as a reference antibody for PF4 recognition, wherein

Note: the PF4 screening factor was calculated based on the anti-HPF 4 antibody AUCs obtained in the reference experiment because there was no human form of anti-HPF 4.

The results of experiment E1 and reference experiment R1 are shown in fig. 20A and 22A and tables 9A and 9B.

Example 3.2: determination of cross-reactivity with platelet factor 4 in human plasma via sandwich ELISA

The same reagents and procedures for reagent preparation as used in example 3.1 were used except:

human plasma spiked with human PF4 (7.3 mg/ml; Molecular Innovation catalog number HPF 4; stored at-30 ℃) (human EDTA plasma bank from 4 different donors; stored at-30 ℃) was used instead of cynomolgus monkey plasma. Human plasma stock of HPF4 spiked was prepared as follows.

A) Preparation of human plasma dilutions:

2 ml of human plasma was pooled at 10000gCentrifuge for 10 minutes. 1.58 ml of supernatant was removed and diluted with 3.42 ml PBST + 0.5% BSA (= 1:3.16 dilution). Subsequent addition of 50 μ l of 10 mg/ml trypsin inhibitor H ₂ And (4) O solution. After incubation for 10 minutes at room temperature, the samples were filtered through a 0.22 μm filter (Millipore catalog number SLGS 0250S).

B) Preparation of stock solution of HPF 4:

add 1 μ l of HPF4 to 99 μ l of PBST + 0.5% BSA buffer = 73 μ g/ml.

C) Preparation of human plasma stock from 10ng/ml HPF4 spike:

0.69 μ l of 73 μ g/ml HPF4 stock solution was added to 5 ml of human plasma diluted 1:3.16, resulting in a 10ng/ml HPF4 admixture of human plasma stock solution.

Dilution series preparation, standard plate set-up, experimental procedures and data analysis for sandwich ELISA with HPF4 spiked human plasma were similar to those described in example 3.1 for sandwich ELISA with cynomolgus monkey plasma.

Binding antibody in experiment E2: same as used in experiment E1 in example 3.1

Reference binding antibody in experiment R2: same as used in reference experiment R1 in example 3.1

The results of experiment E2 and reference experiment R2 are shown in fig. 20B and 22B and tables 9A and 9B.

Table 9A: AUC (or total area of peak) calculated from log-transformed data of experiments E1 and E2, depicted in FIGS. 20A and 20B

^1） chim h1G5 wt is an antibody as described in WO 2007/062853, i.e. a monoclonal antibody having a binding affinity for the a β (20-42) globulomer that is greater than its binding affinity for the a β (1-42) globulomer.

^2） The area under the curve was calculated as described in example 3.1.

Table 9B: AUC (or total area of peak) calculated from logarithmic transformation data referenced to experiments R1 and R2, depicted in FIGS. 22A and 22B

^1） chim h1G5 wt is, for example, WO 2007The antibody described in/062853, i.e., a monoclonal antibody having a binding affinity for the A β (20-42) globulomer that is greater than its binding affinity for the A β (1-42) globulomer.

^2） The area under the curve was calculated as described in example 3.1.

Example 3.3: determination of cross-reactivity with platelet factor 4 in cynomolgus monkey plasma via alignment sandwich ELISA

The reagents and alignment antibodies described in example 3.1 were used anti-mouse IgG (Fc specific; produced in goat; Sigma Cat: M3534; 2.3 mg/ml; stored at-20 ℃ for the murine binding antibody in reference experiment R3) and anti-human IgG (Fc specific; produced in goat; Sigma Cat: I2136; 2.2 mg/ml; stored at-20 ℃ for the human, humanized and human/mouse chimeric binding antibodies in experiment E3).

Methods used in the preparation of the reagents:

blocking solution, primary antibody and TMB solution were prepared as described in example 3.1:

each alignment antibody was diluted to 10 μ g/ml in coating buffer.

Binding antibody in experiment E3: same as used in experiment E1 in example 3.1

Reference binding antibody in experiment R3: the same as used in reference experiment R1 in example 3.1.

Each binding antibody was diluted to 10 μ g/ml (stock) with PBST + 0.5% BSA buffer, and dilution series were prepared as follows:

cynomolgus monkey plasma:

400 mul of cynomolgus monkey plasma bank was set at 10000gCentrifuge for 10 minutes. 158 μ l of supernatant was taken and diluted with 684 μ l PBST + 0.5% BSA (= 1:3.16 dilution). Followed by addition of 10 μ l of 10 mg/ml trypsin inhibitor H ₂ And (4) O solution. After incubation for 10 minutes at room temperature, the samples were passed through a 0.22 μm filter (Millipore mesh)Record SLGS 0250S). Then 500 μ l of this 1:3.16 diluted plasma sample was diluted again 1:31.6 with 15.3 ml PBST + 0.5% BSA buffer, resulting in a total dilution of 1: 100.

Labeling reagent:

the anti-rabbit-POD conjugate lyophilisate was reconstituted in 0.5 ml water. 500 μ l of glycerol was added and 100 μ l aliquots were stored at-20 ℃ for further use. The concentrated labeling reagent was diluted in PBST buffer. The dilution factor was 1: 5000. The reagent was used immediately.

The binding antibody panel set for experiment E2. Dilution of bound antibody. It should be noted that each concentration of each bound antibody was run in duplicate.

The binding antibody plate set up for reference experiment R3. Dilution of bound antibody. It should be noted that each concentration of each bound antibody was run in duplicate.

The procedure used was:

1. 100 μ l of each alignment antibody solution (anti-human IgG for experiment E3; anti-mouse IgG for reference experiment R3) was applied per well and incubated overnight at 4 ℃.

3. 265 μ l blocking solution/well was added and incubated at room temperature for 2 hours.

5. After dilution series preparation of each binding antibody, 100 μ l/well of these antibody dilutions were applied to the plates. The plates were incubated at room temperature for 2 hours.

6. The antibody solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

7. 100 μ l of cynomolgus monkey plasma 1:100 dilution/well was added and incubated for 2 hours at room temperature.

8. The plasma solution was discarded and the wells were washed three times with 250 μ l PBST buffer.

9. 100 μ l primary antibody solution/well was added and incubated at room temperature for 1 hour.

10. The primary antibody solution was discarded, and the wells were washed three times with 250 μ l PBST buffer.

11. 200 μ l of labeling reagent/well was added and incubated for 1 hour at room temperature.

12. Labeling reagents were discarded and wells were washed three times with 250 μ l PBST buffer.

13. 100 μ l of TMB solution was added to each well.

14. The plate color was monitored during development (5-15 minutes at ambient temperature) and when the appropriate color had developed, the reaction was stopped by adding 50 μ l/well of stop solution.

15. The absorbance was read at 450 nm.

Data analysis was performed as described in example 3.1 for the sandwich ELISA with cynomolgus monkey plasma, except that not the plasma dilution factor but the amount of antibody (expressed in ng/ml) was used as X value and thus the concentration effect curve was calculated. Accordingly, the X-value and OD of the logarithmic conversion are based on data of non-curve fitting in the measurement range (final antibody concentration of 10 ng/ml-10000 ng/ml) _450nm Values, area under the curve were determined.

The results of experiment E3 and reference experiment R3 are shown in fig. 21A and 23A and tables 10A and 10B.

Example 3.4: determination of cross-reactivity with platelet factor 4 in human plasma via alignment sandwich ELISA

The same reagents and procedures for reagent preparation as used in example 3.3 were used except:

each alignment antibody for experiment E4 was diluted to 10 μ g/ml in coating buffer and each alignment antibody for experiment E4 was diluted to 50 μ g/ml in coating.

Human plasma spiked with human PF4 (7.3 mg/ml; Molecular Innovation catalog No. HPF 4; stored at-30 ℃) (human EDTA plasma bank from 4 different donors; stored at-30 ℃) was used instead of cynomolgus monkey plasma. Human plasma stock of HPF4 spiked was prepared as follows.

A) Preparation of human plasma dilutions:

4 ml of human plasma was pooled at 10000gCentrifuge for 10 minutes. 3.16 ml of supernatant was removed and diluted with 6.84 ml PBST + 0.5% BSA (= 1:3.16 dilution). Subsequent addition of 100 μ l of 10 mg/ml trypsin inhibitor H ₂ And (4) O solution. After incubation for 10 minutes at room temperature, the samples were filtered through a 0.22 μm filter (Millipore catalog number SLGS 0250S). 5 ml of this 1:3.16 diluted plasma sample was then diluted again 1:3.16 with 10.8 ml PBST + 0.5% BSA buffer, resulting in a total dilution of 1: 10.

B) Preparation of stock HPF 4:

add 1 μ l HPF4 to 99 μ l PBST + 0.5% BSA buffer = 73 μ g/ml.

C) Preparation of human plasma stock from 10ng/ml HPF4 spike:

1.64 μ l of 73 μ g/ml HPF4 stock solution was added to 12 ml of 1:10 diluted human plasma, resulting in a 10ng/ml HPF4 spike in human plasma stock solution.

Preparing a dilution series of the binding antibody; combining the antibody plate; the blocking solution, primary antibody, reagent and TMB solution were prepared as in example 3.3.

Alignment antibody and binding antibody in experiment E4: the same as used in experiment E3 in example 3.3.

Reference to the aligned and bound antibodies in experiment R4: the same as used in reference experiment R3 in example 3.3.

The experimental procedure (but using diluted human plasma at 1:10 in step 7) and data analysis for the aligned sandwich ELISA with HPF4 spiked human plasma was similar to that described in example 3.3 for the aligned sandwich ELISA with cynomolgus monkey plasma.

The results of experiment E4 and reference experiment R4 are shown in fig. 21B and 23B and tables 10A and 10B.

Table 10A: AUC (or total area of peak) calculated from log-transformed data of experiments E3 and E4, depicted in FIGS. 21A and 21B

^2） The area under the curve was calculated as described in example 3.3.

^3） For antibodies 4D10hum #2 and hIgG1, HPF4 binding activity was so low that AUC was calculated to be 0. Thus, the HPF4/aA β antibody ratio was not calculated and indicated as>158 (highest ratio achieved by another antibody (4D 10hum # 1) in this assay).

Table 10B: AUC (or total area of peak) calculated from logarithmic transformation data of reference experiments R3 and R4 as depicted in FIGS. 21A and 21B

^1） m1G5 is an antibody as described in WO 2007/062853, i.e. a monoclonal antibody having a binding affinity for the A β (20-42) globulomer which is greater than its binding affinity for both A β (1-42) globulomers.

^2） The area under the curve was calculated as described in example 3.3.

Sequence listing

<110> Abbott Laboratories

<120> amyloid-beta binding proteins

<130> 10419USO1

<160> 48

<170> PatentIn version 3.5

<210> 1

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa can be Ser or Thr

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa can be Leu or Pro

<220>

<221> misc_feature

<222> (41)..(41)

<223> Xaa can be Phe or Leu

<220>

<221> misc_feature

<222> (42)..(42)

<223> Xaa can be Gln or Leu

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa can be Arg or Lys

<220>

<221> misc_feature

<222> (50)..(50)

<223> Xaa can be Arg or Gln

<400> 1

Asp Val Val Met Thr Gln Xaa Pro Leu Ser Leu Pro Val Thr Xaa Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Xaa Xaa Gln Xaa Pro Gly Gln Ser

35 40 45

Pro Xaa Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 2

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa can be Ile or Val

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa can be Ala or Val

<220>

<221> misc_feature

<222> (29)..(29)

<223> Xaa can be Val or Leu

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa can be Val or Leu

<220>

<221> misc_feature

<222> (49)..(49)

<223> Xaa can be Ser or Gly

<220>

<221> misc_feature

<222> (67)..(67)

<223> Xaa can be Phe or Leu

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa can be Arg or Lys

<220>

<221> misc_feature

<222> (76)..(76)

<223> Xaa can be Asn or Ser

<220>

<221> misc_feature

<222> (78)..(78)

<223> Xaa can be Leu or Val

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Xaa Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Xaa Ser Gly Phe Thr Xaa Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa

35 40 45

Xaa Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Xaa Thr Ile Ser Xaa Asp Asn Ser Lys Xaa Thr Xaa Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 3

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa can be Gln or Glu

<220>

<221> misc_feature

<222> (27)..(27)

<223> Xaa can be Gly or Phe

<220>

<221> misc_feature

<222> (29)..(29)

<223> Xaa can be Ile or Leu

<220>

<221> misc_feature

<222> (37)..(37)

<223> Xaa can be Ile or Val

<220>

<221> misc_feature

<222> (48)..(48)

<223> Xaa can be Ile or Leu

<220>

<221> misc_feature

<222> (67)..(67)

<223> Xaa can be Val or Leu

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa can be Val or Lys

<220>

<221> misc_feature

<222> (76)..(76)

<223> Xaa can be Asn or Ser

<220>

<221> misc_feature

<222> (78)..(78)

<223> Xaa can be Phe or Val

<400> 3

Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Xaa Ser Xaa Ser Ser Tyr

20 25 30

Gly Val His Trp Xaa Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Xaa

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Xaa Thr Ile Ser Xaa Asp Thr Ser Lys Xaa Gln Xaa Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 6

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 7

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 8

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 9

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 9

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 10

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<400> 10

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 11

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 11

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 12

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 12

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 13

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 13

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 14

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 14

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 15

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 15

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 16

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 16

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 17

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 17

Ser Tyr Gly Val His

1 5

<210> 18

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 18

Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met Ser

1 5 10 15

<210> 19

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 19

Asn Ser Asp Val

1

<210> 20

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 20

Lys Ser Ser Gln Ser Leu Leu Asp Ile Asp Gly Lys Thr Tyr Leu Asn

1 5 10 15

<210> 21

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 21

Leu Val Ser Lys Leu Asp Ser

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 22

Trp Gln Gly Thr His Phe Pro Tyr Thr

1 5

<210> 23

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 23

Gln Val Gln Leu Lys Gln Ser Gly Pro Ser Leu Ile Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 24

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 24

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 25

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Phe Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 26

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 26

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 27

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 27

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 28

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<400> 28

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 29

<211> 43

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 29

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala Thr

35 40

<210> 30

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 30

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

<210> 31

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 31

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

<210> 32

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 32

Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn

1 5 10 15

Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala

20 25 30

<210> 33

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 33

Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met

1 5 10 15

Val Gly Gly Val Val Ile Ala

20

<210> 34

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser

20 25 30

<210> 35

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 35

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 36

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 36

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<400> 37

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 38

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<400> 38

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser

20 25 30

<210> 39

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 39

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10

<210> 40

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 40

Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 41

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 41

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 42

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 42

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

20

<210> 43

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 43

Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr

1 5 10 15

<210> 44

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 44

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210> 45

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 45

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

1 5 10

<210> 46

<211> 442

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

180 185 190

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 47

<211> 442

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 47

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

180 185 190

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 48

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of

<400> 48

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An anti-a β (20-42) globulomer antibody comprising:

the heavy chain variable region shown by SEQ ID NO 6 or SEQ ID NO 10 and the light chain variable region shown by SEQ ID NO 14.

2. The antibody of claim 1, wherein the antibody is selected from the group consisting of: immunoglobulin molecules, monoclonal antibodies, scFv, chimeric antibodies, CDR-grafted antibodies, diabodies, humanized antibodies, multispecific antibodies, Fab, DVD, Fab ', F (ab') ₂ And Fv.

3. The antibody of claim 1, wherein the antibody is a disulfide-bonded Fv.

4. The antibody of claim 1, wherein the antibody is a dual specificity antibody.

5. The antibody of claim 1, wherein the antibody is a bispecific antibody.

6. The antibody of any one of claims 1-5, further comprising an immunoglobulin light chain constant region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 27 and SEQ ID NO 28.

7. The antibody according to any one of claims 1-5, wherein the antibody further comprises an agent selected from the group consisting of: (ii) an immunoadhesion molecule; imaging agents and therapeutic agents.

8. The antibody according to any one of claims 1-5, wherein the antibody has a human glycosylation pattern.

9. An isolated nucleic acid encoding the antibody of any one of claims 1-8.

10. A vector comprising the isolated nucleic acid of claim 9.

11. A host cell comprising the vector of claim 10.

12. A method of producing an antibody comprising culturing the host cell of claim 11 in a culture medium under conditions sufficient for production of the antibody.

13. An antibody produced according to the method of claim 12.

14. A pharmaceutical composition comprising the antibody of any one of claims 1-8 or 13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, further comprising at least one additional therapeutic agent.

16. A composition for releasing an antibody, the composition comprising:

(a) a formulation, wherein the formulation comprises the antibody of any one of claims 1-8 or 13 and an additional component, wherein the antibody is crystalline; and

(b) at least one polymeric carrier.